Pharmacological techniques for the in vitro study of the uterus

During the first decade of the 21st century it is unlikely that the pages of specialist pharmacology journals will overflow with papers reporting studies on the isolated uterus. This statement can be made with full confidence and without the aid of either a crystal ball or an advanced degree in bioinformatics. Part of the reason, of course, is the current trend away from tissue-based experimentation and toward cell-based or cell-free systems. But even when this allowance is made the uterus still turns out to be the poor sister among the isolated tissues in the pharmacologist's armamentarium. This is mainly because it has acquired a bad reputation as a difficult tissue to work with (Marshall, 1973a) and that investigators are reluctant to return to a preparation that has previously caused them to have nightmares (Crankshaw, 1990). Indubitably, the isolated uterus does present more technical and interpretational problems than many other smooth muscles. However, the advent of inexpensive laboratory-based computer systems has reduced these difficulties to a minimum. The uterus has a rich history in experimental biology and a broad pharmacology. It is thus an ideal preparation for the study of the complexities of the interrelationships among different signaling systems. Perhaps the time has come for a renaissance.

Important contributions to our current knowledge of physiology and pharmacology have arisen from the use of the isolated uterus since at least the early 1900s. In what follows I present a highly personalized overview of the major milestones. The history of the isolated uterus as an experimental tool is linked inextricably with the neurohypophyseal hormone oxytocin. Dale (1906) made the accidental discovery that neurohypophyseal extracts caused stimulation of the mammalian uterus while he was studying the actions of ergot in intact cats. Three years later (Dale, 1909) he published a systematic study showing stimulatory effects of neurohypophyseal extracts on the uteri of cats, dogs, guinea pigs, rats, and rabbits in vitro as well as in vivo. The result was confirmed independently in the same year by Ott and Scott (1909) using cat and rabbit isolated uteri. The extremely rapid introduction of posterior pituitary extract into obstetrical practice by Blair Bell (1909) necessitated the development of reliable methods for standardization of the oxytocic principle. The first such method utilized the guinea pig isolated uterus (Dale & Laidlaw, 1912). Much later Holton developed the definitive uterine bioassay for oxytocin employing the rat isolated uterus (Holton, 1948). Since that time numerous studies have utilized the isolated uterus in attempts to understand the mechanism of action of oxytocin, to unravel aspects of its physiology, or to develop new drugs that may be useful tocolytics (e.g., Krejci & Polacek, 1968, Crankshaw, 1987, Manning et al., 1995).

The isolated uterus has also played an important part in establishing current concepts and complications of heterologous regulation of cellular signaling pathways. Again Dale made the first important observations. He showed that hypogastric nerve stimulation contracted the pregnant cat's uterus but relaxed the nonpregnant cat's uterus (Dale, 1906). Cushney (1906) found an exactly opposite phenomenon in rabbits. The mechanism of the pregnancy-induced reversal of the response to hypogastric nerve stimulation and to exogenous catecholamines was thoroughly investigated by Marshall and her coworkers (Marshall, 1973b) who showed that the phenomenon could be mimicked by treatment of animals with gonadal steroids (Miller & Marshall, 1965). This work paved the way for the elegant studies of Roberts and colleagues, who demonstrated the role of estrogen in regulating α-adrenoceptor-mediated responses in rabbit myometrium. Isolated myometrium from ovariectomized rabbits and from ovariectomized rabbits treated with 17β-estradiol responded similarly to acetylcholine, whereas concentration-effect curves to noradrenaline were left-shifted in estrogen-treated animals compared to controls. Furthermore, responses to the COMT-resistant, reuptake-insensitive α-adrenoceptor agonist methoxamine were also left-shifted by estrogen (EC₅₀ 23.8 μM in untreated animals and 2.6 μM in treated). The in vitro contractility studies were complemented by radioligand binding studies that showed that estrogen treatment increased the number of α-adrenoceptors expressed by rabbit myometrium but had no effect on β-adrenoceptor expression. Thus, a specific role for estrogens as heterologous regulators of α-adrenoceptor expression was established (Roberts et al., 1981). However, α-adrenoceptor concentrations were later shown to return to pretreatment levels upon the withdrawal of estrogen, whereas enhanced sensitivity persisted (Riemer et al., 1987). These results suggested that estrogen has important regulatory effects on postreceptor processes and subsequent studies confirmed this to be the case (Roberts et al., 1989). In the series of experiments just described, carefully designed and well-executed studies with the isolated uterus played a key role in understanding an important process in modern molecular pharmacology. The lesson that the relationship between receptor number and response can be complex, even confounding, was well taught, but may not have been well learned.

Perhaps the most significant contribution that the isolated uterus has made to pharmacology comes from the work of Kurzrok and Lieb (1930), who demonstrated that components of human seminal fluid could modulate the contractility of the human isolated uterus. This observation led to the discovery of the prostaglandins (Bergström et al., 1968).

Contractile activity of the myometrium (the smooth muscle component of the uterus) is modulated by a broad array of agents often acting in a species-dependent manner. There is no contemporary, authoritative, and comprehensive review of the pharmacology of the myometrium to which the reader can be guided for further information. Consequently in Table 1 I list the major receptors through which modulation of myometrial contractility has been demonstrated using the isolated preparation; the mechanism(s) may be direct or indirect. The references supplied are not intended to be exhaustive or definitive nor do they necessarily represent the original work. Rather they are meant to be as recent as progress in the field allows and to provide a useful starting point for readers interested in further pursuing the topic. I only list receptors for which there is evidence for what I will call “functional expression,” by which I mean that action at the receptors concerned results in a change in the contractile state of the uterus, either stimulation or inhibition of contraction. Regrettably, a number of uterine receptor systems have been characterized using radioligand binding or mRNA expression techniques alone. Receptors may be present in the uterus, but not on myometrial smooth muscle cells. Alternatively they may be present on myometrial smooth muscle cells but not coupled to the regulation of contractility. Thus, binding and mRNA studies might indicate that receptors are expressed in the uterus in the absence of functional expression. For this reason, results from such studies have been ignored.

In addition to the effects mediated through the pharmacologically well-defined receptor systems shown in Table 1, isolated uteri from some species are inhibited by Ca²⁺-channel blockers Kaya et al., 1998, Kaya et al., 1999, ATP-sensitive K⁺-channel openers Cheuk et al., 1993, Okawa et al., 1999, Ca²⁺-dependent K⁺-channel openers Khan et al., 1998, Okawa et al., 1999, nitric oxide Bradley et al., 1998, Okawa et al., 1999, and the polypeptide hormone relaxin (Hughes & Hollingsworth, 1996).

Experimenters who approach the isolated uterus for the first time are faced with a number of confounders that they might not have encountered in work with other, more boring tissues. None of these confounders is unique to the isolated uterus, but they do seem to take on greater proportions in this tissue and to interact with one another in complex ways. When beginning a new series of experiments, it behooves investigators to think carefully about what they are trying to achieve, examine the possible effect of each of the confounders on the outcome, and design their experiments accordingly. That being said, it is not always possible to control all the variables that one would like to control. Critics, invariably those who have never worked with the tissue themselves, are often too ready to dismiss very well-conducted studies on these grounds. Such attitudes do a great disservice to progress in this field. The points here are: (1) that there is still a great deal to be learned from relatively simple experiments; (2) all experiments, no matter what the methods used, need to be interpreted within certain constraints; and (3) that so long as the constraints are recognized and clearly spelled out, new knowledge can be gained.

Major confounders arise because: (1) there are significant interspecies differences; (2) the uterus is a complex organ consisting of different functional cell types; (3) uterine behavior is affected by hormonal status and pregnancy; and (4) the isolated uterus develops spontaneous contractile activity that can make analysis of drug-induced effects rather difficult. In the following section I expand upon these issues with the aim of giving readers sufficient information for them to be able to make the most appropriate choice of experimental conditions to suit their purpose.

Despite the general success of “animal models,” every pharmacologist who attempts to extrapolate information across species runs the risk of making a serious error. There are sufficient instances where a receptor in one species has a significantly different pharmacology from its homologue in another species (e.g., Adham et al., 1992, Burgess et al., 2000). The experimenter needs to be aware of this danger, but it is a generic problem, not restricted to the uterus.

Functional expression of receptors in the uterus is extremely idiosyncratic. A given receptor may be present in one species and appear to be of considerable physiological importance, only for that same receptor to be totally absent in another species. This phenomenon is most graphically illustrated by the prostanoid receptors (Coleman et al., 1990). Table 2 shows the functional expression of prostanoid receptors regulating myometrial contractility among different species. A convincing explanation for the wide diversity of uterine prostanoid receptor expression among species has not been offered. However, the phenomenon has led to some false conclusions. The two synthetic analogues of PGF_2α, now known as cloprostenol and fluprostenol, were initially thought to be selective leuteolytic agents devoid of uterotonic activity (Dukes et al., 1974). This arose from the fact that the guinea pig was used to assess the uterotonic potency of the compounds. Although it was not known at the time, the guinea pig myometrium lacks FP receptors, and cloprostenol and fluprostenol are very selective FP receptor agonists (Coleman et al., 1994), consequently the compounds were not uterotonic. But as can be seen from Table 2, most other species, and particularly humans, do possess myometrial FP receptors. In humans the compounds are potent uterotonics Senior et al., 1992, Senior et al., 1993, the apparent luteolytic selectivity disappears, and along with it two compounds that had enormous potential in human medicine. Clearly, as far as effects of compounds on uterine contractility are concerned, what is true in one species is not necessarily true in another. When embarking on a new program it might be prudent to assume the worst.

Species differences also extend to the anatomy of the uterus. Most polytocous species (e.g., pig, rabbit, rat) have bicornate uteri with the two clearly defined horns joining just above the cervix uteri to form a Y-shaped organ. The uteri of most monotocous species (e.g., human, sheep), on the other hand, exhibit a simpler structure where if horns do exist they are rudimentary, with the bulk of the organ being comprised of the body or corpus uteri. Undoubtedly, these differences in uterine structure between species relate to different physiological functions in nurturing and delivering either many or few fetuses simultaneously. These factors should be borne in mind when choosing the appropriate species for a program of pharmacological investigation of uterine contractility.

Uteri of all eutherian mammals are composed of at least three layers: the endometrium that lines the lumenal surface, the smooth muscle layer or myometrium, and the outer serosa.

The endometrium is the site at which implantation occurs, and through which the conceptus obtains nourishment. The nature and thickness of the endometrial layer is species-dependent, influenced by the menstrual or estrus cycle, and undergoes growth and intense vascularization with pregnancy. Endometrium is composed of a number of different cell types and is an active, secretory structure that is known to release a number of substances that can affect the activity of the smooth muscle layer, including prostanoids (Harper et al., 1991), platelet activating factor (Kudolo & Harper, 1995), and endothelin (Maggi et al., 1991). There may well be more endometrially derived contracting or relaxing factors awaiting discovery. Thus the experimenter interested in uterine contractility must pay particular attention to the endometrium when designing and interpreting experiments. The endometrium may be removed, effects of known endometrial factors may be negated either by preventing their release or blocking their actions, or the endometrium may be left intact if knowledge of the total effect of a drug on the uterus is required. In the later case, of course, the site of action of the drug cannot be guaranteed without further experiment.

By virtue of its preponderant smooth muscle content the myometrium is responsible for force development and contraction. The arrangement of the smooth muscle fibers within the myometrium is species-dependent. Most polytocous species (of which the rat is a particularly good example) have clearly defined longitudinal and circular muscle layers. In the longitudinal layer the majority of muscle cells are oriented with their long axes parallel to the length of the uterine horn. The longitudinal layer is adjacent to the serosa. The circular muscle layer lies between the endometrium and the longitudinal muscle layer, and the majority of its smooth muscle cells have their long axes oriented perpendicular to the length of the uterine horn. Thus, unopposed shortening of the longitudinal layer shortens the length of the uterus, whereas unopposed shortening of the circular muscle constricts the lumen. At the interface some fiber bundles cross from one layer to the next and both blood vessels and nerve terminals are enriched. Often, when intact strips of myometrium are mounted in a longitudinal orientation, the contractions recorded are considered to originate from the longitudinal layer Crankshaw, 1987, Crankshaw & Gaspar, 1992, Niiro et al., 1998 and when they are mounted in circular orientation contractions are ascribed to the circular layer Crankshaw, 1987, Crankshaw & Gaspar, 1992. In some species, however, the two layers are relatively easy to separate and study in isolation (Kitazawa et al., 2000a). Differences in the responsiveness of the two layers are frequently observed (e.g., Tuross et al., 1987, Crankshaw, 1987, Leroy et al., 1991, Haynes et al., 1993, Kitazawa et al., 1997, Isaka et al., 2000, Kitazawa et al., 2000a). Human uterus is a classic example of a case where circular and longitudinal myometrial layers are not clearly defined. Although the majority of the muscle fibers close to the serosal surface tend to run in the longitudinal direction whereas the majority close to the endometrial surface are circular, there is a great deal of heterogeneity at any given site, with numerous oblique fibers. So much so that when the maximal responses and sensitivities to the prostanoid TP receptor antagonist U46619 are compared, strips of human nonpregnant myometrium cut longitudinally from the subserosal layer cannot be distinguished from strips cut longitudinally from the subendometrial layer or from strips cut in the circular orientation (Senchyna & Crankshaw, 1999). There are, however, gradients of responsiveness for other compounds that depend on whether the outer or the inner layer is studied (Leroy et al., 1991). It is erroneous to speak of longitudinal and circular muscle layers when referring to the human uterus. Strips of human uterus isolated from the upper uterine segment respond differently to some prostanoids than do strips isolated from the lower uterine segment (Wikland et al., 1984). There might be a general principle of ovarian/cervical differences in uterine sensitivity to a number of agents, but this has not been explored adequately.

The serosal layer is not known currently to influence the pharmacology of uterine contraction, which of course does not mean that it is inert. In some cases it is easy to remove the serosa, so that any influences it might have can be eliminated; in other cases, removal is impractical and the serosa is ignored.

Gross morphology, behavior, and responsiveness of the uterus vary with the stage of the menstrual or estrus cycle in a manner that suggests they are regulated by the relative concentrations of estrogens and progesterone. While the type and extent of changes are to some degree species-dependent, some generalizations hold across species: uteri taken during estrus, the estrogen-dominated state, are hyperemic, hyperplastic, and hypertrophied in comparison to progesterone-dominated uteri; estrogen-dominated uteri show increased spontaneous activity and increased excitability. The effects of sex steroids on the responses of rabbit uterus to α-adrenoceptor stimulation have already been discussed; responses to several other agonists are also regulated by sex steroids (e.g., Nissenson et al., 1978, Tonoue, 1981, Potvin & Varma, 1990, Gillman & Pennefather, 1998). Unless a given response has been shown categorically not to be affected by hormonal status, it might be appropriate to assume that it likely is. The same is true with pregnancy. Changes in the hormonal milieu during pregnancy are more complex than during the menstrual or estrus cycles, and there is the additional influence of the conceptus(es) to consider. Consequently, the effect of pregnancy on the pharmacology of the isolated uterus is itself often complex and, in the majority of cases, poorly understood. Nevertheless, responses to a number of agonists have been shown to change with pregnancy, although the precise time course is not always clear (e.g., Tuross et al., 1987, Crankshaw, 1987, Potvin & Varma, 1990, Crankshaw & Gaspar, 1992, Kim et al., 1995, Cox et al., 1996, Engstrom et al., 1997).

Of all the confounders that make the isolated uterus such an interesting and challenging tissue to work with, none, I suspect, has caused such cumulative hair-tearing as the phenomenon of spontaneous contractile activity. Isolated uteri develop spontaneous contractile activity whenever they are set up in organ baths. The time taken for spontaneous contractions to appear and their frequency and maximal force are species-, muscle layer-, and hormonal status-dependent. When conventional, noncumulative techniques are used, spontaneous contractile activity can severely interfere with attempts to quantify the effects of drugs on the myometrium because it is often difficult to distinguish a drug-induced contraction from a spontaneous contraction. For this reason a great deal of ingenuity has been used in efforts to make the isolated myometrium behave like a decent (vascular?) smooth muscle. The favored techniques are to lower the temperature and to reduce the concentrations of either Ca²⁺ or Mg²⁺, or both, in the physiological salt solution. A number of classic solutions have been devised for specific purposes van Dyke & Hastings, 1928, Garcia de Jalon et al., 1945, Munsick, 1960 and have been used with variable degrees of success by numerous investigators. However, messing with extracellular ion concentrations can have unpredictable effects on pharmacology (Krejčı́ & Poláček, 1968) and is not recommended, particularly for the investigation of novel compounds or receptor systems.

So what is to be done? As it turns out, when isolated uteri are set up in a manner that is appropriate for the particular species and allowed adequate equilibration time, the spontaneous activity becomes regular and predictable. The spontaneous activity in these circumstances does not interfere significantly with the ability to detect drug-induced effects provided that cumulative additions are made, strict protocols (particularly with respect to time) are followed, and appropriate parameters of the response are measured Wainman et al., 1988, Dyal & Crankshaw, 1988, Crankshaw & Gaspar, 1995, Kitazawa et al., 2000b, Popat & Crankshaw, 2001. For the latter provision, the availability of online data digitization and collection has been a boon, which early investigators of uterine contractility in vitro were unfortunate to miss.