Myometrial characteristics of the syrian hamster uterine smooth muscle cell line,SHM

Summary We describe several characteristics of a novel smooth muscle cell line, SHM (Syrian hamster myometrium) derived from a primary uterine leiomyosarcoma which was induced by chronic estrogen plus androgen treatment of a female Syrian (golden) hamster. To determine the usefulness of the SHM cell line as a model for understanding myometrial function and its regulation, we have examined the morphologic and immunocytochemical properties of these cells, and the ability of uterotonic agonists to activate transmembrane signaling via phosphoinositide hydrolysis. The SHM cells exhibited a spindle-shape, smooth musclelike morphology when subconfluent, and a more compact, stellate shape at confluence. Like primary myocytes, SHM cells expressed the intermediate filament desmin and the contractile protein alpha smooth muscle actin, but not the epithelial antigen cytokeratin. Norepinephrine and bradykinin, which stimulate contraction and inositol polyphosphate production in the uterus, also stimulated inositol polyphosphate production in SHM cells. The maximal phosphoinositide signaling responses were lower in SHM cells compared with primary hamster uterine myocytes. We conclude that the SHM cell line exhibits primary uterine myocyte characteristics, and may therefore be a useful system for examining the mechanisms through which myometrial functions are regulated.     

Morphological changes induced in mouse mammary gland by porcine and human relaxin

The effects of pure porcine relaxin and of human decidual extracts with relaxin-like activity on the mammary gland of virgin mice primed with estrogen have been studied by the light microscope. Porcine relaxin enhanced the changes induced by estrogen alone; the effect was different in the various mammary tissues. In the stroma, relaxin only slightly increased the loosening of connective tissue, the extent of the adipose tissue and of the capillary bed, as well as the degranulation of the mast cells. The changes in the parenchyma, such as elongation and branching of ducts, are strikingly enhanced. Moreover, relaxin seems to promote differentiation of the cells forming the walls of distal ducts, and of the myoepithelial cells. Tissue extracts of human decidua with relaxin-like activity induce changes in the mammary gland similar to those due to porcine relaxin. Such data indicate that relaxin synergizes with estrogen to cause growth of ducts of the mammary gland and that tissue extracts of human decidua have a similar effect, thus providing further evidence that decidua may be a source of relaxin in humans.     

Nitric oxide as the regulator of intracellular homeostasis in the uterus myocytes

The published data on the mechanisms and regulation of active and passive Ca2+ transport in the myometrium have been analyzed. Particular attention is paid to the cGMP-dependent and independent pathways of action of nitric oxide or its derivatives on intracellular Ca2+ homeostasis of uterine smooth muscle and its contractile activity. Information on the effect of nitric oxide on Ca2+ -transport systems of other types of smooth muscles is provided in a comparative aspect. Based on own experimental results and literature data a scheme of NO action in the myometrium is suggested in which nitric oxide or its derivatives cause Ca2+ -dependent polarization of the sarcolemma. In accordance with our results, this effect may be based on the increase of sarcolemma Ca2+ permeability under the influence of NO or its derivatives and the stimulation of at least the initial passive transport of the cation in the myocytes mediated by dihydropyridine-sensitive channels. Additional factors that contribute to the polarization of the membrane are the increase of protons transport from the muscle cells and stimulation of Na+, K+ -ATPase. Acting on the sarcoplasmic reticulum, nitrosactive compounds activate the inclusion of calcium in this compartment and inhibit Ca2+ -induced release of the cation. The latter effects are able to provide compensation for NO-induced Ca2+ increase in myocytes and supress the electromechanical coupling at Ca2+ release from the reticulum. NO-derivates also inhibit a key link in the smooth muscle contractile act--the formation of the Ca2+ -calmodulin complex.     

Effects of active substances fromStaphylococcus aureus (protein a and peptidoglycan) on neurotransmitter-induced contractions of smooth muscles

Modulatory effects of protein A (PA) and peptidoglycan (PG) fromStaphylococcus aureus on the smooth muscle contractions elicited by neurotransmitters were studied. PA and PG were found to suppress the myometrium smooth muscle contractions elicited by acetylcholine applications. The effects of the substances on the contractions were due to non-competitive inhibition through an enhancement of the potassium-dependent conductance of the smooth muscle cell membrane. PA and PG did not affect the myometrium contractions elicited by oxytocin, or the contractions of the coronary arteries elicited by acetylcholine or noradrenaline.Neirofiziologiya/Neurophysiology, Vol. 28, No. 1, pp. 30–35, January–February, 1996.     

Effect of ethanol on ATP-hydrolase activity of myometrial actomyosin

In order of estimating some regularities of ethanol effective action on the uterus smooth muscles contractile proteins the effects of spiritus introduced into incubation medium on myometrium actomyosine ATP-hydrolase activity and superprecipitation was studied. ATP-hydrolase activity was displayed as more sensitive to ethanol action; its dependence on ethyl spiritus concentration had three-phase character expressed in two inhibiting and one activating sites. While defining the kinetic parameters of ATP hydrolysis reaction catalysed by uterus myometrium the correlation between inhibition the ATP-hydrolase activity of myometrium contractile complex under introducing into incubation medium 2% ethanol and decreasing the affinity of actomyosine to Mg2+ was made; the highest activating effect of ethanol on ATP-hydrolase activity of actomyosine complex in the presence of 8% ethanol correlated with increasing the affinity of actomyosine to Mg2+ and ATP.     

The effects of herbal oxytocics on the isolated "stripped" myometrium model

Decoctions of Agapanthus africanus and Clivia miniata are used as oxytocic agents in South African traditional herbal medicine. Aqueous extracts of A. africanus and C. miniata leaves have been shown to possess similar uterotonic activities in the isolated whole uterus preparation. The uterus however, comprises a myometrial and an endometrial layer and the activity of both oxytocin and the prostaglandins differs in these layers. The aim of this study was to determine the uterotonic activity of the herbal remedies in an endometrium-free preparation (i.e. "stripped" myometrium) and, if active, whether this effect could be related to prostaglandin synthesis or to interaction with specific receptors. The effects of the herbal extracts were tested on the isolated "stripped" rat myometrium preparation. Both herbal extracts caused a direct contractile response by the isolated tissue. Pretreatment of the myometrium with either plant extract augmented the initial response to acetylcholine. Preincubation with atropine inhibited the response to cumulative dosage of Agapanthus extract but had no effect on the response to Clivia. Indomethacin administration did not affect the response of the myometrium to cumulative dosage of acetylcholine, oxytocin or Clivia extract but inhibited the response to Agapanthus extract. These results clearly indicate that the Agapanthus and Clivia herbal extracts exhibited uterotonic activity in this model. The study illustrates that the "stripped" myometrium model has successfully differentiated between the mechanisms of action of two herbal oxytocics compared to the whole uterus preparation where their uterotonic activity was thought to be similar.     

Effect of polyamines on actomyosine-ATP-ase activity in smooth muscles of the uterus

The ability of the aliphatic polyamines to inhibit [figure: see text] the ATPase activity of smooth muscle actomyosine satisfies the succession: spermine > spermidine > putrescine that is correlated with magnitude of positive charge at physiological value of pH. The most effective inhibitor of the ATP hydrolysis process is the spermine, which highest inhibitory action is manifested at 10(-3) M concentration, in lesser concentration (10(-5) M) activates the actomyosine ATPase. While defining the kinetic parameters of the ATP hydrolysis reaction catalyzed by uterus myometrium the correlation between inhibiting the ATPase activity of myometrium contractile complex under introduction into the incubation medium of 10(-3) M spermine and decreasing the affinity of actomyosine for ATP was made; the activating effect of spermine on ATPase activity of actomyosine complex in the presence of 10(-5) M spermine correlated with the increase of actomyosine affinity for Mg2+.     

Immunomorphological identification of myoepithelial cells in mixed tumors of the mammary gland in dogs

Myoepithelial cells (MC) in mixed tumors of the mammary gland in dogs were identified with the Coons indirect method with the aid of monospecific antiserum to smooth muscle myosin. In 2 of 5 observations, anaplastic carcinoma and adenocarcinomas were detected in the presence of a mixed tumor. Immunochemical study of the myoepithelium demonstrated varying fluorescence intensity and ununiform pattern of MC distribution in tumor tissue of the mammary gland. MC were detected in lobular structures in the form of islet accumulations or diffuse vegetations from cells with a poorly fluorescent rim of the cytoplasm. In the ducts with papillary epimyoepithelial proliferations, MC occupied the peripheral position, showing bright fluorescence of the cytoplasm and hypertrophied processes. The fluorescence intensity decreased as MC displaced towards the stroma or lumen of the ducts. The changeability of specific staining of the myoepithelium may attest to different levels of cell differentiation. The presence of immature forms of MC in mixed tumors is likely to be due to the modulatory character of cell differentiation and is determined by the totality of factors that apparently play the triggerring part in derepression of the genes of specific synthesis of smooth muscle proteins in undifferentiated cells of the epithelium of the terminal ducts and alveoli.     

T-type Ca2+ channels in non-vascular smooth muscles

T-type Ca2+ current has been recorded in smooth muscle myocytes, and associated interstitial cells, isolated from the gastro-intestinal tract, urinary bladder, urethra, prostate gland, myometrium, vas deferens, lymphatic vessels and airways smooth muscle. By contrast, current through such channels has not been recorded from other tissues, such as the ureter. Whilst the properties of this Ca2+ current are similar in most of these cells, with respect to their voltage-dependence, ion selectivity and response to channel modulators, some differences have been recorded, most notably in the gastro-intestinal tract, and may demand a reappraisal of how a T-type Ca2+ current is characterised. The functions of such a current in different tissues remains uncertain. In most of smooth muscles discussed in this review, it is hypothesised that it underlies rhythmic or spontaneous electrical activity, especially in concert with other current-carrying systems, such as Ca2+-activated outward currents. Of equal interest is that the T-type Ca2+ channel may be a target for agents that modulate tissue function, especially in pathological conditions, or are the site of secondary effects of agents used in clinical medicine. For example, T-type Ca2+ channel modulators have been proposed to reduce overactive muscular activity in the gastro-intestinal or urinary tract, or function as tocolytic agents: and the action of volatile anaesthetics on them in airways smooth muscle requires consideration in their overall action.     

Reactivity of the smooth muscle tissue of the large intestine under conditions of experimental obstruction]

Morphohistochemical and autoradiographic techniques were used to study the reactivity of the large intestine tunica mascularis under experimental ileus. The smooth muscle cells are subjected to pronounced changes. At early terms (1-2 days) reactive-compensatory reactions are observed. They include increased metabolic processes, hypertrophy of myocytes, their increased synthetic and proliferative activity. Simultaneously there appear degenerative phonomena which become predominant on the 4th-6th days. The level of metabolism in the smooth muscle tissue drops and the synthetic and proliferative activity of myocytes decreases. They undergo atrophy and lyse.     

Connexin43 in porcine myocardium and non-pregnant myometrium

It is generally accepted that connexin43 (Cx43) is a major constituent of heart and myometrial gap junctions. However, the presence of Cx43 gap junctions in non-pregnant myometrium is still poorly documented. Tissue sections of porcine heart and non-pregnant uterus and myometrial smooth muscle cell cultures were immunostained with monoclonal antibody against Cx43. In the heart, intensive immunostaining was confined to the intercalated discs as previously reported. In the non-pregnant uterus, punctuate immunostaining of Cx43 was seen throughout the myometrium along cell interfaces between myocytes. The expression of Cx43 was sustained in cultured smooth muscle cells isolated from non-pregnant myometrium. Western blotting has detected single isoform of Cx43 in both, cardiac and myometrial tissues. The electrophoretic mobility of porcine heart Cx43 was similar to that of myometrial isoform but different from the pattern of mobility of Cx43 of the rat heart. Hence, porcine myometrium may provide attractive model for studying cellular mechanisms triggering expression of gap junction protein in normal (non-pregnant) uterus.     

Localization of NTPDase1/CD39 in normal and transformed human pancreas.

Elevated levels of extracellular ATP have been observed in many tumors. We have localized NTPDase1/CD39, one of the principal extracellular nucleotide-hydrolyzing enzymes, in normal and cancerous human pancreas. NTPDase/E-ATPDase activity was demonstrated with an enzyme histochemical technique on cryosections of human pancreas. Acinar and duct epithelial cells were devoid of E-ATPDase activity in both normal and transformed tissue. Endothelial cells and smooth muscle around blood vessels and larger ducts showed strong activity. Nerves, connective tissue, and the beta-cells of the islets were also stained. In cancerous tissue this activity was diminished in the smooth muscle around the ducts and was absent from newly formed connective tissue. Immunostaining for CD39 supported these results but revealed the presence of inactive CD39 in the duct epithelial cells. We hypothesize that the significantly diminished activity of NTPDase1 in the tissues surrounding the ducts may be associated with the processes that lead to tumor formation in human pancreas.     

The mechanism of calcium control of smooth muscle tonic contraction

It has been shown in the experiments carried out on a fraction of inverted vesicles of myometrium sarcolemma that ATP-dependent Ca2+ transport system prevents dissipation of the calcium gradient directed from the intervesicular space outward with subsequent establishment of the stationary level of cation content inside the membrane vesicles (a blocker of electro-controlled calcium channels diltiasems was present in the incubation medium). Ortovanadatean inhibitor of the sarcolemma calcium pump suppressed Ca2+ stationary exchange in the vesicles fraction. The value of calcium stationary content in the vesicle membrane was regulated both by a change of the calcium pump activity (by varying Mg2+ concentration in the ATP-containing incubation medium), and by modification of calcium permeability of the vesicles (by varying concentration of ionophore A-23187 in this medium). In the presence of diltiasem and ortovanadate the Ca2+ basal current entering the myocytes from hyperpotassium washing solution activated the smooth muscle tonic contraction. In the absence of ortovanadate no contractile response was observed. On the basis of the evidence obtained a mechanism of calcium control of myometrium tonic contraction is proposed. According to this mechanism the Ca2+ current entering the unexcited myocytes under physiological conditions is efficiently compensated by the calcium pump of the sarcolemma. The inhibition of the latter (or an increase of the sarcolemma basal calcium permeability) provides further slow transition of the stationary value of Ca2+ concentration in the myoplasm to a new higher level and activation of the smooth muscle contraction accordingly.     

A kinetic model of stationary calcium metabolism in smooth muscle cells

A model is proposed for Ca2+ stationary exchange in the myometrium cells in the absence of effects activating calcium channels of the plasmic membrane. The results of the model analysis point to an important role of the calcium pump (but not on Na(+)-Ca2+ exchanger) of the sarcolemma in providing the long-term regulation of physiologically significant concentration of ionized calcium in the smooth muscle cells. Ability of the calcium pump to efficiently compensate Ca2+ basal current continuously entering the myocytes at rest is proved. It is suggested that the stationary transsarcolemmal exchange of calcium (the system "basal calcium current--ATP-dependent transfer of Ca2+") underlies the control mechanism of the myometrium basal tonus, while a disturbance of the stationary state (with the pump inhibition) provides activation of the smooth muscle tonic contraction.     

Characterization of the tissue‐level Ca2+ signals in spontaneously contracting human myometrium

In the labouring uterus, millions of myocytes forming the complex geometrical structure of myometrium contract in synchrony to increase intrauterine pressure, dilate the cervix and eventually expel the foetus through the birth canal. The mechanisms underlying the precise coordination of contractions in human myometrium are not completely understood. In the present study, we have characterized the spatio‐temporal properties of tissue‐level [Ca²⁺]_i transients in thin slices of intact human myometrium. We found that the waveform of [Ca²⁺]_i transients and isotonic contractions recorded from thin slices was similar to the waveform of isometric contractions recorded from the larger strips in traditional organ bath experiments, suggesting that the spatio‐temporal information obtained from thin slices is representative of the whole tissue. By comparing the time course of [Ca²⁺]_i transients in individual cells to that recorded from the bundles of myocytes we found that the majority of myocytes produce rapidly propagating long‐lasting [Ca²⁺]_i transients accompanied by contractions. We also found a small number of cells showing desynchronized [Ca²⁺]_i oscillations that did not trigger contractions. The [Ca²⁺]_i oscillations in these cells were insensitive to nifedipine, but readily inhibited by the T‐type Ca²⁺ channel inhibitor NNC55‐0396. In conclusion, our data suggest that the spread of [Ca²⁺]_i signals in human myometrium is achieved via propagation of long‐lasting action potentials. The propagation was fast when action potentials propagated along bundles of myocytes and slower when propagating between the bundles of uterine myocytes.     

Immunohistochemistry using an antibody to unphosphorylated connexin 43 to identify human myometrial interstitial cells

Background

Myometrial smooth myocytes contract as a result of electrical signalling via a process called excitation-contraction coupling. This process is understood in great detail at the cellular level but the generation and coordination of electrical signals throughout the myometrium are incompletely understood. Recent evidence concerning the vital role of interstitial cells of Cajal in tissue-level signalling in gastrointestinal tract, and the presence of similar cells in urinary tract smooth muscle may be relevant for future research into myometrial contractility but there remains a lack of evidence regarding these cells in the myometrium.

Methods

Single stain immunohistochemical and double stain immunofluorescence techniques visualised antibodies directed against total connexin 43, unphosphorylated connexin 43, KIT, alpha-SMA and prolyl 4-hydroxylase in myometrial biopsies from 26 women representing all stages of reproductive life.

Results

Myometrial smooth myocytes from term uterine biopsies expressed connexin 43 in a punctate pattern typical of gap junctions. However, on the boundaries of the smooth muscle bundles, cells were present with a more uniform staining pattern. These cells continued to possess the same staining characteristics in non-pregnant biopsies whereas the smooth myocytes no longer expressed connexin 43. Immunohistochemistry using an antibody directed against connexin 43 unphosphorylated at serine 368 showed that it is this isoform that is expressed continually by these cells. Double-stain immunofluorescence for unphosphorylated connexin 43 and KIT, an established marker for interstitial cells, revealed a complete match indicating these cells are myometrial interstitial cells (MICs). MICs had elongated cell processes and were located mainly on the surface of the smooth muscle bundles and within the fibromuscular septum. No particular arrangement of cells as plexuses was observed. Antibody to prolyl 4-hydroxylase identified fibroblasts as separate from MICs.

Conclusion

MICs are identified consistently on the boundaries of smooth muscle bundles in both the pregnant and non-pregnant uterus and are distinct from fibroblasts. The uniform distribution of connexin 43 on the cell membrane of MICs, rather than localisation in gap junction plaques, may represent the presence of connexin hemichannels. This antibody specificity may aid future study of this potentially important cell type.     

Characterization of oxytocin receptors in the uterus and mammary gland.

High affinity binding sites for 3[H] oxytocin have been demonstrated in particulate fractions from rat uterus and oviduct, myometrium from the sow, ewe and human, ewe endometrium, and mammary gland from the lactating rat. The binding activity has been localized to enriched plasma membrane fractions from the rat uterus and mammary gland; cells isolated from the mammary gland also bind oxytocin. The apparent dissociation constant (Kd) for the interaction of oxytocin with its binding sites in a variety of tissue preparations is in the nanomolar range. The concentration of oxytocin eliciting half-maximal contraction of the rat isolated uterus corresponds to the apparent Kd of oxytocin interaction with uterine particulate fractions. Binding is specific with respect to the target tissue or cell, as well as to the ligand. The affinity of binding sites for oxytocin analogues corresponds generally to their potencies as agonists or antagonists. Factors that affect the binding of oxytocin affect the biological response in the same way. For example, certain divalent metal ions, which increase oxytocin binding activity, enhance the sensitivity of the contractile response of the uterus and mammary gland to oxytocin. Estrogen administration, which increases the uterine binding of oxytocin, increases the sensitivity of the myometrium to oxytocin. The myometrium binds the most oxytocin at estrus and is most sensitive to oxtocin at that time. The dgree of stimulation by oxytocin of prostaglandin F2alpha synthesis by ewe endometrium is paralleled by an increased concentration of oxytocin binding sites. The marked increase in sensitivity to oxytocin of the rat uterus occurring on the day of parturition also is reflected by the amount of oxytocin bound by the uterus. Because of the many correlations between oxytocin binding and bioactivity, it appears that oxytocin binding sites on the plasma membrane of target cells constitute the recognition part of oxytocin receptors.     

Age-related changes in the epithelial and stromal compartments of the mammary gland in normocalcemic mice lacking the vitamin D3 receptor

The vitamin D(3) receptor (VDR) serves as a negative growth regulator during mammary gland development via suppression of branching morphogenesis during puberty and modulation of differentiation and apoptosis during pregnancy, lactation and involution. To assess the role of the VDR in the aging mammary gland, we utilized 12, 14, and 16 month old VDR knockout (KO) and wild type (WT) mice for assessment of integrity of the epithelial and stromal compartments, steroid hormone levels and signaling pathways. Our data indicate that VDR ablation is associated with ductal ectasia of the primary mammary ducts, loss of secondary and tertiary ductal branches and atrophy of the mammary fat pad. In association with loss of the white adipose tissue compartment, smooth muscle actin staining is increased in glands from VDR KO mice, suggesting a change in the stromal microenviroment. Activation of caspase-3 and increased Bax expression in mammary tissue of VDR KO mice suggests that enhanced apoptosis may contribute to loss of ductal branching. These morphological changes in the glands of VDR KO mice are associated with ovarian failure and reduced serum 17β-estradiol. VDR KO mice also exhibit progressive loss of adipose tissue stores, hypoleptinemia and increased metabolic rate with age. These developmental studies indicate that, under normocalcemic conditions, loss of VDR signaling is associated with age-related estrogen deficiency, disruption of epithelial ductal branching, abnormal energy expenditure and atrophy of the mammary adipose compartment.     

Uterine myometrial ferritin and blood plasma transferrin as natural modulators of Ca-calmodulin-independent cyclic nucleotide phosphodiesterase from cow uterine myometrium

The iron storage protein ferritin was purified to electrophoretic homogeneity from cow uterine myometrium. Its Mr did not exceed 440, 000 and H-chains predominated in the subunit composition; the iron saturation was 43 iron ions per protein molecule. The uterine myometrial ferritin was a potent natural modulator of Ca-calmodulin-independent phosphodiesterase (Ca-CM-independent PDE, EC 3.1.4.17) isolated from the same tissue. Addition of iron-poor ferritin from uterine myometrium and iron-reach liver ferritin caused three- and two-fold inhibition of the enzyme activity, respectively. The iron transport protein transferrin in iron-saturated and iron-depleted forms can also inhibit Ca-CM-independent PDE activity by two-fold. In both cases, the degree of saturation with iron was not crucial for the inhibitory effects of these proteins on the enzyme activity. These data suggest that iron homeostasis proteins can modulate the cyclic nucleotide level in non-nervous tissue via interaction with enzymes involved in cyclic nucleotide hydrolysis.