kao

Ionic channel functions of mammalian smooth muscles
Our research efforts are focused on the physiology and pharmacology of mammalian smooth muscles and on tetrodotoxin and saxitoxin. The two foci are tied together by a common thread of underlying ionic channel functions. Our work is primarily based on electrophysiological methods, using voltage-clamped small multicellular preparations, single skeletal muscle fibers, single freshly dissociated smooth myocytes, or single channel events in membrane patches.

Smooth muscles comprise a diverse group of involuntary excitable tissues, which are widely dispersed throughout the body, subserving important physiological functions and involved in major diseases. Because of difficulties posed by the small individual smooth muscle cells, their ionic channel functions have not been studied in earnest until the recent introduction of the tight-seal patch-clamp method and successful regimens of isolating individual myocytes. We are interested in ionic channel functions of visceral smooth muscles, including those of the intestine, uterus, and the lower urinary tract. Because we have studied them in small non-enzyme-treated multicellular preparations, single freshly dissociated myocytes, and as single channels in membrane patches, we can relate events and/or processes at single channel molecules to physiological functions and pharmacological responses at tissue levels.

b-adrenergic effects on intestinal and uterine smooth muscles.

Activation of b₂ receptors in these tissues causes relaxation (inhibition in classical pharmacology). The basis of the action is an increased open probability of a class of large-conductance K⁺ channels. By use of specific mimetic and/or blocking agents, we have delineated the sequence of steps, on single-channels and on whole-cells, as G_s protein --> adenylate cyclase -- > cyclic AMP -- > protein kinase A. The final step most probably involves phosphorylation of maxi-K channels.

The increased membrane conductance induced by b-agonist would reduce excitability by requiring more current to elicite action potentials (hence, the "inhibition"). Although b-agonists work well on intestinal smooth muscles, their effects on pregnant uterine muscle are limited. Clinical uses of b-agonists to arrest premature uterine contractions have led to transient tocolysis, but have not altered the ultimate outcome of premature births. Preterm labor and its consequences are the major neonatal public health issue because of the associated high infant mortality and long-term morbidity as well as the high cost of treatment. From our studies, we find that maxi-K channels are limited in pregnant uterine myocytes through a combination of altered expression conditions, and a reduction in their relative contribution to the whole-cell K⁺ current, suggesting an essential futility of using b-agonists for the management of premature uterine contractions (the most common current form of tocolytic therapy). To address this pressing public health issue, we believe drug management of preterm labor needs a new approach.

Cardiac antiarrhythmic agents as potential uterine tocolytics.

In uterine myocytes, we find a high-affinity type tetrodotoxin-sensitive Na⁺ current which appears upon estrogen stimulation, and gradually increases relative to a co-existing Ca²⁺-channel, as pregnancy progresses towards term. This Na⁺current is an unusual phenotype for smooth muscles, which generally depend on voltage-gated Ca²⁺ channels only for the inward current. Coupled with our reservations about the utility of b- adrenergic agonists for uterine tocolysis, we find commonly used class 1B cardiac antiarrhythmic agents, lidocaine, mexiletine and tocainide to be effective in blocking the myometrial Na⁺ channel. Thus, the possibility exists that these familiar FDA-approved drugs may serve as uterine tocolytics.

B. Tetrodotoxin (TTX) and Saxitoxin (STX).

These toxins are important tools because of their potent and selective blocking actions on the voltage-gated Na⁺-channel. They were pivotal in the isolation of channels, and the ultimate recombinant identification of the primary structures of Na⁺ channels.

We have been involved in the early clarification of the actions of these toxins, in the recognition that the chemically different toxin molecules produced identical effects, and in identifying the common structural features responsible for their actions. To move forward in using these toxins to help understand the structure of the Na⁺ channel, we have synthesized a specifically labelled ³H-tetrodotoxin of high specific activity and a non-exchangeable marker (US patent 5,288,870). We have also made a photoactivatable adduct of tetrodotoxin which may help us to locate the tetrodotoxin-binding site.

In addition to their utility as important investigative tools, both TTX and STX (especially the latter) are of public-health significance because of their involvement in episodic outbreaks of food poisoning.

Selected Publications

Kao, C. Y., and Levinson, S. R. (1986). Tetrodotoxin, saxitoxin and the molecular biology of the sodium channel. Annals of the New York Academy of Sciences, Vol. 497. (With references to other work).

Fan, S. F., Wang, S. Y., and Kao, C. Y. (1993). The tranduction system in the isoproterenol activation of the Ca2⁺-activated K⁺ channel in guinea pig taenia coli myocyte. J. Gen. Physiol. 102, 257-275.

Kao, C. Y., and Wang, S. Y. (1994). Effects of lidocaine on rat myometrial sodium channels and implications for the management of preterm labor. American Journal of Obstetrics and Gynecology 171, 446-454.

Kao, C. Y., and Carsten, M. E., eds. (1997). Cellular Aspects of Smooth Muscle Function. Cambridge University Press.

Yoshino, M., Wang, S. Y., and Kao, C. Y. (1997). Sodium and calcium inward currents in freshly dissociated smooth myocytes of rat uterus. J. Gen. Physiol. 110, 565-577.

Figure 1. Probable binding conformations of tetrodotoxin (1a) and saxitoxin (1b). In spite of dissimilar structures, toxins bind to same sites (a - g) on sodium channel, sharing one ion-pairing site (a) via their guanidinium moieties, and three hydrogen-bonding sites (b, c, f), in addition to other sites. Such binding accounts for their high potency, ready reversibility, and similar biological actions.

Figure 2. Inward currents in single smooth myocyte from late-pregnant rat uterus, and effects of lidocaine. In trace C (control) initial downward deflection is sodium current followed by slower calcium current. Rat myocytes can serve as models, because similar channels are seen in pregnant human myocytes. 0.1 mM lidocaine selectively blocks sodium current (trace L).