Myometrial interstitial cells and the coordination of myometrial contractility.

A strict regulation of contractility in the uterus and fallopian tube is essential for various reproductive functions. The uterus contributes, through either increased contractility or periods of relative quiescence, to: (i) expulsion of menstrual debris, (ii) sperm transport, (iii) adequate embryo placement during implantation, (iv) enlarging its capacity during pregnancy and (v) parturition. The dominant cell population of the uterine wall consists of smooth muscle cells that contain the contractile apparatus responsible for the generation of contractile force. Recent interest has focused on a new population of cells located throughout the myometrium on the borders of smooth muscle bundles. These cells are similar to interstitial cells of Cajal (ICC) in the gut that are responsible for the generation of electrical slow waves that control peristalsis. A precise role for myometrial Cajal-like interstitial cells (m-ICLC) has not been identified. m-ICLC express the c-kit receptor, involved in creating and maintaining the ICC phenotype in the gastrointestinal tract. However, both acute and prolonged inhibition of this receptor with the c-kit antagonist imatinib mesylate does not appear to affect the spontaneous contractility of myometrium. Calcium imaging of live tissue slices suggests that contractile signalling starts on the borders of smooth muscle bundles where m-ICLC are located and recently the possible role of extracellular ATP signalling from m-ICLC has been studied. This manuscript reviews the evidence regarding tissue-level signalling in the myometrium with a particular emphasis on the anatomical and possible functional aspects of m-ICLC as new elements of the contractile mechanisms in the uterus.

Interstitial Cajal-like cells in human uterus and fallopian tube.

Recently, parallels have been drawn between enteric interstitial cells of Cajal (ICC) and similar cells outside the gut-interstitial Cajal-like cells (ICLC). This article reviews our laboratory findings on ICLC in the female reproductive tract. Since the morphology and function of ICLC are still a subject of debate, our purpose was to investigate whether ICLC are present in the fallopian tube and/or uterus, and if they share ultrastructural and immunohistochemical (IHC) features and/or functional roles. We studied ICLC presence in the human fallopian tube and myometrium primarily by light microscopy, and then by transmission electron microscopy (TEM), in tissue samples and at a single cell level. Taking advantage of our ICLC studies of several organs (pancreas, mammary gland, myocardium), we assembled a set of criteria, derived from ultrastructural features of ICLC, called "platinum standard." Besides the putative pacemaker function, ICLC might have other physiological roles, depending on tissue type (e.g., intercellular signaling, immune surveillance, steroid sensors). Consequently, there is a great urge for a conceptual framework that could allow a better understanding, from a functional point of view, and more so, as the ICLC processes are the longest cellular prolongations (except neurons).

Interstitial Cajal-like cells (ICLC) as steroid hormone sensors in human myometrium: immunocytochemical approach.

Expression of estrogen (ER) and progesterone (PR) receptors was investigated in cultured human normal myometrial cells (non-pregnant uterus, fertile period). The ER and PR expression was studied by immunohistochemistry and immunofluorescence on either myocytes or interstitial Cajal-like cells (ICLC). Only those cells double immunostained for c-kit and steroid receptors were considered as ICLC. ER and/or PR immunoreactivity was localized in ICLC, primarily concentrated at the nucleus level, but it was also observed in the cell body (cytoplasm) and processes. Stronger immunopositive reaction in the ICLC nucleus for PR than for ER was noted. Under our experimental conditions, a clear positive repeatable reaction for steroid receptors could not be detected in myocytes. In conclusion, these data suggest that ICLC could be true hormonal 'sensors', possibly participating in the regulation of human myometrial contractions (via gap junctions with myocytes and/or by paracrine signaling).

Imatinib inhibits spontaneous rhythmic contractions of human uterus and intestine.

The interstitial cells of Cajal (ICC) in the digestive tract and ICC-like cells in extradigestive organs express the c-kit tyrosine-kinase receptor, and have been implicated as pacemakers of smooth muscle spontaneous activity. We used imatinib mesylate (Glivec) to investigate whether c-kit activity of Cajal-like cells in human myometrium is involved in spontaneous rhythmic contractions of human uterine smooth muscle, taking intestinal smooth muscle as a reference tissue. We show that imatinib concentration-dependently inhibited the myogenic contractions of human myometrium in the organ bath, while it significantly affected noradrenaline or K(+)-induced contractions only at concentrations exceeding 50 muM. An inhibitory antibody directed against the extracellular domain of the platelet derived growth factor receptor (PDGFR), another target of imatinib that is expressed by the uterine muscle cells themselves, failed to affect myogenic contractions. These results suggest that Cajal-type cells of human myometrium, as well as ICC of intestinal smooth muscle, participate in myogenic contractile mechanisms, via a novel ligand-independent c-kit/CD117 tyrosine-kinase signaling.

Interstitial Cajal-like cells (ICLC) in atrial myocardium: ultrastructural and immunohistochemical characterization.

We have previously reported (Hinescu & Popescu, 2005) the existence of interstitial Cajal-like cells (ICLC), by transmission electron microscopy, in human atrial myocardium. In the present study, ICLC were identified with non-conventional light microscopy (NCLM) on semi-thin sections stained with toluidine blue and immunohistochemistry (IHC) for CD117/c-kit, CD34, vimentin and other additional antigens for differential diagnosis. Quantitatively, on semi-thin sections, ICLC represent about 1-1.5% of the atrial myocardial volume (vs. approximately 45% working myocytes, approximately 2% endothelial cells, 3-4% for other interstitial cells, and the remaining percentage: extracellular matrix). Roughly, there is one ICLC for 8-10 working atrial myocytes in the intercellular space, beneath the epicardium, with a characteristic (pyriform, spindle or triangular) shape. These ICLC usually have 2-3 definitory processes, emerging from cell body, which usually embrace atrial myocytes (260 nm average distance plasmalemma/sarcolemma) or establish close contact with nerve fibers or capillaries (approximately 420 nm average distance to endothelial cells). Cell prolongations are characteristic: very thin (mean thickness = 0.15+/-0.1 microm), very long for a non-nervous cell (several tens of microm) and moniliform (uneven caliber). Stromal synapses between ICLC and other interstitial cells (macrophages) were found (e.g. in a multicontact type synapse, the average synaptic cleft was approximately 65 nm). Naturally, the usual cell organelles (mitochondria, smooth and rough endoplasmic reticulum, intermediate filaments) are relatively well developed. Caveolae were also visible on cell prolongations. No thick filaments were detected. IHC showed that ICLC were slightly and inconsistently positive for CD117/c-kit, variously co-expressed CD34 and EGF receptor, but appeared strongly positive for vimentin, along their prolongations. Some ICLC seemed positive for a-smooth muscle actin and tau protein, but were negative for nestin, desmin, CD13 and S-100. In conclusion, we provide further evidence of the existence of ICLC in human atrial myocardium, supporting the possible ICLC role in pacemaking, secretion (juxta- and/or paracrine), intercellular signaling (neurons and myocytes). For pathology, ICLC might as well be 'players' in arrhythmogenesis and atrial remodeling.

C-kit immunopositive interstitial cells (Cajal-type) in human myometrium.

Previous reports describing Cajal-like interstitial cells in human uterus are contradictory in terms of c-kit immunoreactivity: either negative (but vimentin-positive) in pregnant myometrium, or positive, presumably in the endometrium. The aim of this study was to verify the existence of human myometrial Cajal-like interstitial cells (m-CLIC). Six different, complementary approaches were used: 1) methylene-blue supravital staining of tissue samples (cryosections), 2) methylene blue and Janus green B vital staining (m-CLIC and mitochondrial markers, respectively), and 3) extracellular single-unit electrophysiological recordings in cell cultures, 4) non-conventional light microscopy on glutaraldehyde/osmium fixed, Epon-embedded semi-thin sections (less than 1 microm) stained with toluidine blue (TSM), 5) transmission electron microscopy (TEM), and 6) immunofluorescence (IF). We found m-CLIC in myometrial cryosections and in cell cultures. In vitro, m-CLIC represented approximately 7% of the total cell number. m-CLIC had 2-3 characteristic processes which were very long (approximately 60 microm), very thin (< or =0.5 microm) and moniliform. The dilated portions of processes usually accommodated mitochondria. In vitro, m-CLIC exhibited spontaneous electrical activity (62.4+/-7.22 mV membrane potentials, short duration: 1.197+/-0.04 ms). Moreover, m-CLIC fulfilled the usual TEM criteria, the so-called 'gold' or 'platinum' standards (e.g. the presence of discontinuous basal lamina, caveolae, endoplasmic reticulum, and close contacts between each other, with myocytes, nerve fibers and/or capillaries etc.). IF showed that m-CLIC express CD117/c-kit, sometimes associated with CD34, with vimentin along their processes. In conclusion, we describe myometrial Cajal-like interstitial cells that have affinity for methylene blue and Janus green B vital dyes, fulfill (all) TEM criteria, express CD117/c-kit and have spontaneous electric activity.

Novel type of interstitial cell (Cajal-like) in human fallopian tube.

We describe here--presumably for the first time--a Cajal-like type of tubal interstitial cells (t-ICC), resembling the archetypal enteric ICC. t-ICC were demonstrated in situ and in vitro on fresh preparations (tissue cryosections and primary cell cultures) using methylene-blue, crystal-violet, Janus-Green B or MitoTracker-Green FM Probe vital stainings. Also, t-ICC were identified in fixed specimens by light microscopy (methylene-blue, Giemsa, trichrome stainings, Gomori silver-impregnation) or transmission electron microscopy (TEM). The positive diagnosis of t-ICC was strengthened by immunohistochemistry (IHC; CD117/c-kit+ and other 14 antigens) and immunofluorescence (IF; CD117/c-kit+ and other 7 antigens). The spatial density of t-ICC (ampullar-segment cryosections) was 100-150 cells/mm2. Non-conventional light microscopy (NCLM) of Epon semithin-sections revealed a network-like distribution of t-ICC in lamina propria and smooth muscle meshwork. t-ICC appeared located beneath of epithelium, in a 10-15 microm thick 'belt', where 18+/-2% of cells were t-ICC. In the whole lamina propria, t-ICC were about 9%, and in muscularis approximately 7%. In toto, t-ICC represent ~8% of subepithelial cells, as counted by NCLM. In vitro, t-ICC were 9.9+/-0.9% of total cell population. TEM showed that the diagnostic 'gold standard' (Huizinga et al., 1997) is fulfilled by 'our' t-ICC. However, we suggest a 'platinum standard', adding a new defining criterion- characteristic cytoplasmic processes (number: 1-5; length: tens of microm; thickness: < or =0.5 microm; aspect: moniliform; branching: dichotomous; organization: network, labyrinthic-system). Quantitatively, the ultrastructural architecture of t-ICC is: nucleus, 23.6+/-3.2% of cell volume, with heterochromatin 49.1+/-3.8%; mitochondria, 4.8+/-1.7%; rough and smooth endoplasmic-reticulum (1.1+/-0.6%, 1.0+/-0.2%, respectively); caveolae, 3.4+/-0.5%. We found more caveolae on the surface of cell processes versus cell body, as confirmed by IF for caveolins. Occasionally, the so-called 'Ca2+-release units' (subplasmalemmal close associations of caveolae+endoplasmic reticulum+mitochondria) were detected in the dilations of cell processes. Electrophysiological single unit recordings of t-ICC in primary cultures indicated sustained spontaneous electrical activity (amplitude of membrane potentials: 57.26+/-6.56 mV). Besides the CD117/c-kit marker, t-ICC expressed variously CD34, caveolins 1&2, alpha-SMA, S-100, vimentin, nestin, desmin, NK-1. t-ICC were negative for: CD68, CD1a, CD62P, NSE, GFAP, chromogranin-A, PGP9.5, but IHC showed the possible existence of (neuro)endocrine cells in tubal interstitium. We call them 'JF cells'. In conclusion, the identification of t-ICC might open the door for understanding some tubal functions, e.g. pace-making/peristaltism, secretion (auto-, juxta- and/or paracrine), regulation of neurotransmission (nitrergic/purinergic) and intercellular signaling, via the very long processes. Furthermore, t-ICC might even be uncommitted bipotential progenitor cells.

Interstitial cells of Cajal in pancreas.

We show here (presumably for the first time) a special type of cell in the human and rat exocrine pancreas. These cells have phenotypic characteristics of the enteric interstitial cells of Cajal (ICC). To identify pancreatic interstitial cells of Cajal (pICC) we used routine light microscopy, non-conventional light microscopy (less than 1 mum semi-thin sections of Epon-embedded specimens cut by ultramicrotomy and stained with Toluidine blue), transmission electron microscopy (TEM), and immunocytochemistry. The results showed that pICC can be recognized easily by light microscopy, particularly on semi-thin sections, as well as by TEM. Two-dimensional reconstructions from serial photos suggest a network-like spatial distribution of pICC. pICC represent 3.3+/-0.5% of all pancreatic cells, and seem to establish close spatial relationships with: capillaries (43%), acini (40%), stellate cells (14%), nerve fibres (3%). Most of pICC (88%) have 2 or 3 long processes (tens of mum) emerging from the cell body. TEM data show that pICC meet the criteria for positive diagnosis as ICC (e.g. numerous mitochondria, 8.7+/-0.8% of cytoplasm). Immunocytochemistry revealed that pICC are CD117/c-kit and CD34 positive. We found pICC positive (40-50%) for smooth muscle alpha-actin or S-100, and, occasionally, for CD68, NK1 neurokinin receptor and vimentin. The reactions for desmin and chromogranin A were negative in pICC. At present, only hypotheses and speculations can be formulated on the possible role of the pICC (e.g., juxtacrine and/or paracrine roles). In conclusion, the quite-established dogma: "ICC only in cavitary organs" is overpassed.