Papaverine-Induced Inhibition of Electrical and Mechanical Activity and Calcium Movements of Rat Ileal Smooth Muscle

Papaverine, the major benzylisoquinoline alkaloid of opium, possesses potent relaxatory action on a number of smooth muscles (Santi, Ferrari & Contessa, 1964; Diamond & Marshall, 1969; Tashiro & Tomita, 1970; Bauer & Capek, 1971; Poch & Kukovetz, 1971; Bauer, Kadlek & Seferna, 1974; Reinhardt, Wagner & Schumann, 1974; Uruno et al. 1974; Miyamoto et al. 1976). For a number of years, papaverine has been employed as a vasodilator agent because of its relaxatory effect on vascular smooth muscle (Kukovetz & Poch, 1970; Triner et al. 1971) and as an antiarrhythmic agent on the heart (Reinhardt et al. 1977; Whipple, 1977). However, there is no concensus of opinion regarding the mechanism of papaverine-induced relaxation of these various visceral muscles.

There is evidence that papaverine inhibits phosphodiesterases in visceral muscle (Poch & Kukovetz, 1971; Urono et al. 1974; Miyamoto et al. 1976; Takayanagi, Yamashita & Kasuya, 1978), implicating an increase in cell cyclic AMP level as the mediator of the relaxatory mechanism (Triner et al. 1971; Poch & Umfahrer, 1976; Honeyman, Merriam & Fay, 1978) as is thought to be the case in methylxanthine action on smooth muscle (Robison, Butcher & Sutherland, 1971 ; Aberg & Andersson, 1972; Berridge, 1975; Theobald, Syson & Burrin, 1978). However, the role of cyclic AMP as the secondary regulator of drug-induced contraction and relaxation of smooth muscle is far from clear. A number of inconsistencies in the relationship between inhibition of phosphodiesterases and the relaxatory effect of papaverine have been found (Andersson et al. 1977; Polson et al. 1978).

Although papaverine increased cyclic AMP levels in cardiac muscle and in arterial smooth muscle, no correlation could be found between cyclic AMP level and contractility (Kukovetz & Poch, 1970; Reinhardt et al. 1977). In contrast, papaverine was found to be without effect on cyclic AMP level in seminal vesicle smooth muscle, but caused strong relaxations of KCl-induced contractures (Gamo et al. 1977). In addition, cyclic AMP was not involved in phosphorylase activation nor in the relaxation of uterine smooth muscle caused by papaverine and a number of other relaxants (Diamond, 1973; Diamond & Holmes, 1975). Cyclic GMP has also been implicated in contraction and relaxation of a number of smooth muscles (Schultz et al. 1973; Bulbring & Hardman, 1976; Schultz & Hardman, 1975; Andersson & Djarv, 1978). Some correlation does exist between development of tension and increase in cyclic GMP levels, especially in potassium depolarized muscle (Andersson & Djarv, 1978), although evidence suggests that cyclic GMP may not be involved in contraction and relaxation of taenia coli and uterus in response to catecholamines (Bulbring & Hardman, 1976).

There is, however, one major drawback in implicating cyclic nucleotides in the major immediate effects of papaverine on smooth muscle. The very speed of action of papaverine must cast doubt on direct mediation by the rather circuitous cyclic AMP/cyclic GMP/phosphodiesterase route. The speed of papaverine-induced responses could well be due to a much more direct action on the muscle, possibly at some stage in the early excitation-contraction coupling sequence, although this possibility has not been investigated.

In this study, the direct actions of papaverine on electrical and mechanical activity of ileal smooth muscle have been examined, and attempts have been made to correlate these actions with calcium influx and efflux. The most immediate actions of papaverine on this smooth muscle seem more related to an action on calcium influx than on cyclic nucleotide effects (Saad & Huddart, 1979).

Wistar-strain albino rats were used throughout this study. The animals were killed by a blow to the head, the ileum was removed immediately and placed in aerated Krebs saline at 37 °C. This saline contained (in mM): NaCl, 120·7; KC1, 5·9; CaCl₂, 2·5; MgCl₂, 1·2; Na₂HPO₄, 1·2; NaHC0₃, 15·5 and glucose, 11·5 at pH 7·3. A concentrated stock solution of papaverine in Krebs saline was prepared, and appropriate amounts were added directly to the organ bath, efflux salines and incubaion media. For studies in calcium-free media, calcium was omitted, but chelating gents such as EDTA and EGTA were not employed in order to prevent undesirable artificial removal of intracellular free Ca by these agents.

For tension recording, 2 cm ileal strips were mounted vertically in a 50 ml organ bath and were connected to a Washington Instruments isotonic strain gauge which fed into a curvilinear pen recorder. For simultaneous recording of tension and membrane potentials, preparations were mounted horizontally in an organ bath. Membrane potentials were recorded with glass capillary intracellular microelectrodes filled with 3 M-KCI. Signals were fed via a high-impedance probe for display on a Tektronix 502 A dual-beam oscilloscope and they were recorded on a Bell & Howell ultraviolet oscillograph connected in parallel with the oscilloscope. Preparations were continuously aerated throughout the experiments.

The techniques for estimating ⁴⁵Ca influx and efflux of ileal smooth muscle strips have been described in detail elsewhere (Huddart & Syson, 1975; Huddart & Saad, 1977, 1978). Mitochondria and membrane vesicles (microsomes) of ileal smooth muscle were prepared for “Ca uptake studies as follows. Sections of ileal muscle were homogenized in ice-cold extraction medium (100 mM-KCl and 1 mM imadazole, pH 6·8) using a Teflon coaxial homogenizer. The homogenate was first centrifuged at 1000 g for 30 min to remove cell debris and the mitochondria were sedimented from the supernatant by centrifugation for 30 min at 8000 g. Microsomes were sedimented from the supernatant by centrifugation for 60 min at 28000g. All centrifugations were carried out at 2 °C. The mitochondrial and microsomal pellets were resuspended in extraction medium and adjusted to 1 mg protein/ml following biuret analysis.

To determine ⁴⁵Ca uptake by mitochondria and microsomes, 1 ml of suspension was mixed with 1 ml of incubation medium containing (in mM): MgCl₂, 4; CaCl_a, 2·5; ATP, 2; tris maleate buffer, 40 at pH 7·0 with 0·1 µCi ⁴⁵Ca/ml. The reaction proceeded at room temperature and was stopped at various time intervals by passage through membrane filters of pore size 0·45 µm. The suspensions on the filters were washed to remove extraneous ⁴⁵Ca by passing 20 ml of Krebs saline through the filter. After drying, ⁴⁵Ca activity retained on the filters was estimated in a Packard Tricarb liquid scintilation counter. Background ⁴⁵Ca contamination of blank control filters was subtracted from the experimental counts.

In the concentration range 10–150 µM, papaverine caused relaxation of the spontaneous rhythmic contractions of the rat ileum, accompanied by a fall in resting tension (Fig. 1). The concentration threshold for consistently observable relaxation was remarkably low at only 10 µM, and the dose-dependency of papaverine-induced relaxation of this intestinal smooth muscle is shown in Fig. 2. The inhibition of contraction was measured simply as the percentage reduction in height of spontaneous contractions. In addition to altering contractile activity, papaverine also slowed the spontaneous rhythm.

Pre-treatment of ileal smooth muscle with papaverine salines for periods as short a 2 min caused a dose-dependent inhibition of subsequent KCI-induced contracture tension. At 10 µM, the phasic part of the contracture was greatly reduced while the tonic part of the contracture was completely inhibited. At all higher concentrations both the phasic and the tonic KCl contracture were virtually abolished (Fig. 3). To check whether papaverine could influence preparations in which calcium influx had been stimulated, papaverine was applied to muscles induced into contracture with high KCl salines. In such preparations, papaverine was found to strongly inhibit the KCI-induced tonic contracture (Fig. 4). Papaverine inhibition was completely reversible even at the highest concentrations used. All preparations showed a return to normal resting tension and spontaneous contractility on perfusion with control Krebs saline (Fig. 4).

Papaverine, in the concentration range 10–150 µM, caused a decline in spontaneous electrical activity of the ileum. Electrical activity of ileal smooth muscle of the rat consists of phases of slow waves of 8–14 mV amplitude with superimposed spikes of 8–15 mV amplitude (Syson & Huddart, 1976), separated by periods of small slow wave activity only. These events correspond to the cyclical peristalsis of the preparation. It is usually not possible with simultaneous recording to achieve a perfect register of the electrical and mechanical events since electrical activity is recorded from a single cell of the preparation while tension has to be recorded from the whole muscle strip. However, reasonable overall correlation can often be achieved.

Typical spikes and slow waves recorded from the ileum are shown in Fig. 5. At low concentrations of papaverine (10–50 µM) spikes were slowly abolished while some small slow wave activity continued (Fig. 5 a). In this trace, papaverine has clearly inhibited the cyclical burst of slow waves and spikes compared with the previous cyclical burst 60 s earlier. At higher concentrations both spikes and slow waves declined in a matter of 10–20 s, accompanied by a corresponding decline in mechanical activity (Fig. 5 b). The inhibition of spontaneous electrical activity was not accompanied by any significant change in the resting membrane potential. The mean resting potential of this particular smooth muscle was – 41·2 ± 2·3 mV. After 5 min treatment with 50 µM papaverine the resting potential was –39·8 ±3·6 mV. The effect of papaverine on electrical activity was reversible. On return to control Krebs saline, spontaneous slow waves and spikes were slowly restored (Fig. 5 c).

The relationship between inward calcium movement and contractile activation of smooth muscle has been firmly established (Joiner, 1972; Huddart & Syson, 1975; Casteels & Van Breemen, 1975; Barratt & Huddart, 1978). Any agent that blocks inward calcium movement or competes with calcium at surface binding sites impairs normal spontaneous contractile activity (Van Breemen et al. 1972; Reinhardt et al. 1974; Saida, Yanaura & Chujyo, 1974; Godfraind, 1976; Huddart & Saad, 1977; Deth, 1978). The clear inhibition of ileal smooth muscle spikes by papaverine suggested that this agent may block inward calcium movement since spikes are calciumdependent (Job, 1969; Huddart & Hunt, 1975). It was therefore thought pertinent to examine the effect of papaverine on calcium influx into ileal smooth muscle strips.

The results of these experiments are summarized in Fig. 6, where a clear inhibition of calcium uptake of between 30–40% is seen in papaverine-containing media.

It is possible to test whether this level of inhibition of calcium influx by papaverine is of real significance in the excitation-contraction coupling cycle. If ileal smooth muscle preparations are incubated in calcium-free Krebs saline, spontaneous activity is greatly reduced, the rhythm is slowed, and finally abolished. When calcium-containing media are then introduced to these preparations, normal activity is quickly established by rapid calcium influx (Huddart & Saad, 1977, 1978). When such preparations are again exposed to calcium-free media, activity is again reduced and the rhythm slowed (Fig. 7). These results show just how labile contractile activity is in this smooth muscle and how dependent it is on extracellular calcium.

If these experiments are repeated in papaverine-containing media, quite different results are obtained (Fig. 8). In Fig. 8(a), papaverine (0·05 mM) has inhibited spontaneous activity of the ileum maintained in normal calcium media. When extra calcium is added to this preparation in the form of three successive additions of 2 mM calcium (total Ca content 8·5 mM), this still fails to restore normal activity in contrast to the situation seen in Fig. 7. In Fig. 8(b–d), preparations were first exposed to calcium-free media (at first arrow) that caused a reduction in activity, as seen in the experiments in Fig. 7. Papaverine was then added to the preparations at the second arrow. Successive additions of calcium to these preparations at the third and fourth arrows completely failed to restore normal spontaneous activity in the presence of papaverine, in contrast to reintroduction of calcium in the control experiment in Fig. 7, where papaverine was not present. With papaverine present, the ileum failed to respond to KC1 even in high-calcium media (Fig. 8(c), fifth arrow), but when papaverine saline was replaced by normal physiological saline, spontaneous activity was slowly restored (Fig. 8(d), fifth arrow). The reintroduction of calcium to preparations in either normal or calcium-free media failed to restore normal contractile activity as long as papaverine was present. These results strongly suggest that papaverine has inhibited calcium influx to such an extent as to prevent a sufficient inward calcium signal to trigger normal contractions, and they correlate well with the results of ⁴⁵Ca uptake by the ileum in the presence of papaverine. This inhibition of calcium influx by papaverine is very reminiscent of quinine action on smooth muscle (Huddart & Saad, 1977).

To test any actions by papaverine on gross cellular calcium movements, ⁴⁵Ca efflux from ileal smooth muscle strips was examined. The normal “Ca efflux curve of ileal smooth muscle is shown in Fig. 9. When papaverine is added to the efflux saline during the slow, presumably intracellular, efflux phase, a significant inhibition of efflux is seen, from 0·17%/min to about 0·07%/min. This represents a sharp fall in myoplasmic free calcium level in the presence of papaverine. That this fall is almost certainly related to inhibition of calcium influx is confirmed when ⁴⁵Ca efflux in high KCl salines is examined. KCl-induced depolarization of smooth muscle causes a sharp rise in calcium influx (Sperelakis, 1962; Briggs, 1962; Urakawa & Holland, 1964), and this can be seen clearly as a large stimulation of “Ca efflux in Fig. 10, as has previously been shown in this preparation (Huddart & Syson, 1975). In the presence of papaverine this KCl-induced stimulation of efflux is greatly inhibited.

The effects of papaverine so far described on calcium influx and efflux do not rule out additional effects on the subcellular structures involved in calcium binding. Various membrane vesicles (microsomes) and mitochondria have been implicated in calcium regulation during the excitation-contraction coupling cycle of a number of visceral muscles (Huddart & Syson, 1975; Heumann, 1976; Uchida, 1976; Huddart, Hunt & Oates, 1977; Janis & Daniel, 1977; Raeymaekers et al.1977;Nilsson et al, 1978; Theobald et al. 1978). To check for any involvement of these subcellular fractions in papaverine responses of the ileum, calcium uptake by isolated microsomes and mitochondria was investigated. The results of these experiments are summarized in Fig. 11. Papaverine was found to be without effect on calcium uptake by ileal smooth muscle mitochondria, although it caused a very slight stimulation of calcium uptake by the microsomal fraction in vitro.

The confusion surrounding the mechanism of papaverine-induced relaxation of smooth muscle stems from the fact that there has been no single comprehensive study of papaverine action on all stages of the excitation-contraction coupling mechanism in any one muscle.

Tashiro & Tomita (1970) concluded that papaverine decreased mechanical activity by suppressing spontaneous spike activity in the smooth muscle of guinea-pig taenia coli. They claimed that excess calcium (6 mM) could restore mechanical responses but failed to restore electrical activity, a clear inconsistency since it is the spikes which generate thythmic contractions. In the rat ileum studied here, papaverine does indeed suppress spikes and rhythmic contractions, but neither event can be restored by excess calcium in the presence of papaverine. Bauer et al. (1974) found that papaverine inhibited the nicotinic cholinoreceptors of the intramural parasympathetic ganglia of guinea-pig taenia coli and ileum. This observation is certainly consistent with the inhibition of spike activity seen in this preparation.

A number of biochemical investigations have linked papaverine-induced relaxation of smooth muscle with cyclic nucleotide metabolism (Kukovetz & Poch, 1970; Nishikori, Takenaka & Maeno, 1977; Fabiato & Fabiato, 1978; Honeyman et al. 1978). The simple picture portrayed by these studies is that papaverine-induced inhibition of phosphodiesterases causes a rise in cell cyclic AMP which reduces myoplasmic free calcium by a stimulation of intracellular calcium binding, resulting in relaxation. Such a simple picture is now not tenable. A rise in cell cyclic AMP is not a prerequisite for relaxation in a number of smooth muscles (Diamond, 1973; Diamond & Holmes, 1975; Clyman et al. 1976; Gamo et al. 1977; Sands, Mascali & Paietta, 1977).

If the primary action of papaverine in inducing relaxation is mediated via a rise in cyclic AMP, then papaverine should stimulate calcium binding by subcellular fractions such as the mitochondria and microsomes (Kukovetz & Poch, 1970). In the rat ileum the concentrations of papaverine that bring about immediate relaxation are without significant effect on mitochondrial or microsomal calcium uptake, as is also the case in vascular smooth muscle (Sands et al. 1977). In these smooth muscles, at least, any effects of papaverine on intracellular calcium binding are not significant factors in the relaxatory mechanism. These results do not support the view that papaverine’s immediate effect on tension is mediated via cyclic nucleotide metabolism. Direct investigation of cyclic AMP effects on rat ileal smooth muscle also fail to support this simple model (Saad & Huddart, 1979). The incidental observation that papaverine inhibits oxidative phosphorylation in guinea-pig ileum and duodenum (Santi et al. 1964) also provides no satisfactory explanation of the immediate inhibitory effects of papaverine on tension.

There is evidence in the literature that papaverine may directly influence contractility by altering calcium fluxes in smooth muscle. Tomiyama, Takayanagi & Takagi (1973) found that papaverine reduced calcium influx into guinea-pig taenia caecum and relaxed KCl-induced contractures, although they observed a surprisingly anomalous small increase in ⁴⁵Ca efflux. Their overall view was that papaverine action could be explicable in terms of a fall in cell free calcium without involving cyclic AMP.

The evidence from this present study on rat ileum is that the relaxatory actions of papaverine can all be related to its observed inhibition of calcium influx. The inhibition of calcium-dependent spike activity is consistent with this view. The observation that addition of calcium to ileal muscles in calcium-free salines failed to restore contractility if papaverine was present is further evidence that papaverine has inhibited calcium influx.

Studies on isotopic efflux from tissues have to be viewed with some caution since a number of not easily quantifiable complications make apparently simple results open to a number of different interpretations. Isotopic back-flux may occur and intracellular specific activity constantly falls during the course of the efflux experiment as ⁴⁵Ca is lost by replacement with ⁴⁰Ca. The view commonly held from a number of efflux studies is that efflux of calcium is dependent upon the level of calcium within the cell, hence stimulation or inhibition of the slow calcium efflux phase reflects a rise or a fall in cell free calcium (Sperelakis, 1962; Blaustein & Hodgkin, 1969; Deth & Van Breemen, 1977; Deth, 1978). The ⁴⁵Ca efflux response thus provides a test of the effect of papaverine on intracellular free calcium. The consistent inhibition of ⁴⁵Ca efflux by papaverine seen here represents a clear fall in myoplasmic free calcium. The experiments summarized in Fig. 6 show that papaverine inhibits calcium influx and would thus reduce entry of ⁴⁰Ca to the cell. While this may increase cytoplasmic ⁴⁵Ca specific activity, the fall in cell free calcium would greatly reduce ⁴⁰Ca/⁴⁶Ca exchangeability, thus a decline in efflux rate coefficient would be expected. An alternative explanation could be that perhaps papaverine stimulated intracellular calcium binding since this would be reflected as a fall in ⁴⁵Ca efflux rate coefficient. That this explanation is unlikely is indicated by the results summarized in Fig. 11, where papaverine was found to be without effect on mitochondrial calcium binding and to only very slightly stimulate microsomal binding. On balance, the reduction in calcium efflux seen here could not easily be related to stimulation of mitochondrial or microsomal calcium binding but it is perfectly consistent with the observed inhibition of calcium influx by papaverine. This view is strengthened by the observation that KCl-stimulated ^4BCa efflux is also inhibited by papaverine, this being related to an inhibition of the calcium influx which accompanies KCl-induced responses.

Undoubtedly papaverine and a number of other alkaloids do have actions on cell cyclic nucleotide metabolism and on calcium binding and release by sub-cellular calcium storage structures. However, the inconsistency of such actions by papaverine in various smooth muscles, coupled with the immediacy of papaverine action suggests that the most obvious effects of this alkaloid are more consistent with an inhibition of calcium influx which is the trigger stage of the whole excitation-contraction coupling mechanism.

We are most grateful to our colleague Dr S. Hunt for much discussion during the course of this study. Mr Saad was supported by an Iraq Government Research Scholarship during this study.