Endothelial glycocalyx structure in the intact carotid artery: a two-photon laser scanning microscopy study.

BACKGROUND: The endothelial glycocalyx (EG) is the carbohydrate-rich luminal lining of endothelial cells that mediates permeability and blood cell-vessel wall interactions. To establish an atheroprotective role of the EG, adequate imaging and quantification of its properties in intact, viable, atherogenesis-prone arteries is needed. METHODS: Carotid arteries of C57Bl6/J mice (n=22) were isolated including the bifurcation, mounted in a perfusion chamber, and perfused with fluorescent lectin wheat germ agglutinin-fluorescein isothiocyanate. The EG was visualized through the vessel wall using two-photon laser scanning microscopy. An image quantification protocol was developed to assess EG thickness, which was sensitive to hyaluronidase-induced changes. RESULTS: In the lesion-protected common carotid artery, EG thickness was found to be 2.3 ± 0.1 µm (mean ± SEM), while the surface area devoid of (wheat germ agglutinin-sensitive) EG was 8.9 ± 4.2%. Data from the external carotid artery were similar (2.5 ± 0.1 µm; 9.1 ± 5.0%). In the atherogenesis-prone internal carotid artery the EG-devoid surface area was significantly higher (27.4 ± 5.5%, p<0.05); thickness at the remaining areas was 2.5 ± 0.1 µm. CONCLUSION: The EG can be adequately imaged and quantified using two-photon laser scanning microscopy in intact, viable mounted carotid arteries. Spatial EG differences could underlie atherogenesis.

Evaluation of magnetic resonance vessel size imaging by two-photon laser scanning microscopy.

MR vessel size imaging (MR-VSI) is increasingly applied to noninvasively assess microvascular properties of tumors and to evaluate tumor response to antiangiogenic treatment. MR-VSI provides measures for the microvessel radius and fractional blood volume of tumor tissue. However, data have not yet been evaluated with three-dimensional microscopy techniques. Therefore, three-dimensional two-photon laser scanning microscopy (TPLSM) was performed to assess microvascular radius and fractional vessel volume in tumor and muscle tissue. TPLSM data displayed a mazelike architecture of the tumor microvasculature and mainly parallel oriented muscle microvessels. For both MR-VSI and TPLSM, a larger vessel radius and fractional blood volume were found in the tumor rim than in the core. The microvessel radius was approximately six times larger in tumor and muscle for MR-VSI than for TPLSM. The tumor blood volume was 4-fold lower with MR-VSI than with TPLSM, whereas muscle blood volume was comparable for both techniques. Differences between the tumor rim, core, and muscle tissue showed similar trends for both MR-VSI and TPLSM parameters. These results indicate that MR-VSI does not provide absolute measures of microvascular morphology; however, it does reflect heterogeneity in microvascular morphology. Hence, MR-VSI may be used to assess differences in microvascular morphology.

In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy.

In vivo (molecular) imaging of the vessel wall of large arteries at subcellular resolution is crucial for unraveling vascular pathophysiology. We previously showed the applicability of two-photon laser scanning microscopy (TPLSM) in mounted arteries ex vivo. However, in vivo TPLSM has thus far suffered from in-frame and between-frame motion artifacts due to arterial movement with cardiac and respiratory activity. Now, motion artifacts are suppressed by accelerated image acquisition triggered on cardiac and respiratory activity. In vivo TPLSM is performed on rat renal and mouse carotid arteries, both surgically exposed and labeled fluorescently (cell nuclei, elastin, and collagen). The use of short acquisition times consistently limit in-frame motion artifacts. Additionally, triggered imaging reduces between-frame artifacts. Indeed, structures in the vessel wall (cell nuclei, elastic laminae) can be imaged at subcellular resolution. In mechanically damaged carotid arteries, even the subendothelial collagen sheet (approximately 1 microm) is visualized using collagen-targeted quantum dots. We demonstrate stable in vivo imaging of large arteries at subcellular resolution using TPLSM triggered on cardiac and respiratory cycles. This creates great opportunities for studying (diseased) arteries in vivo or immediate validation of in vivo molecular imaging techniques such as magnetic resonance imaging (MRI), ultrasound, and positron emission tomography (PET).

Nanoparticles for optical molecular imaging of atherosclerosis.

Molecular imaging contributes to future personalized medicine dedicated to the treatment of cardiovascular disease, the leading cause of mortality in industrialized countries. Endoscope-compatible optical imaging techniques would offer a stand-alone alternative and high spatial resolution validation technique to clinically accepted imaging techniques in the (intravascular) assessment of vulnerable atherosclerotic lesions, which are predisposed to initiate acute clinical events. Efficient optical visualization of molecular epitopes specific for vulnerable atherosclerotic lesions requires targeting of high-quality optical-contrast-enhancing particles. In this review, we provide an overview of both current optical nanoparticles and targeting ligands for optical molecular imaging of atherosclerotic lesions and speculate on their applicability in the clinical setting.

Two-photon microscopy on vital carotid arteries: imaging the relationship between collagen and inflammatory cells in atherosclerotic plaques.

We used two-photon laser scanning microscopy (TPLSM) to demonstrate for the first time its potential in studying relational details at the cellular level of atherogenesis in intact, viable mouse carotid arteries. Isolated and mounted arteries of ApoE-/-mice, aged 15 or 21 weeks (7 and 13 weeks on western diet), were imaged after labeling with specific fluorescent markers for cell nuclei, inflammatory cells, collagen, and lipids. Data were compared with C57BL6/J mice fed a chow diet. Control vessels had intact endothelium without adhering blood cells or significant intimal collagen labeling. In ApoE-/-mice already at 15 weeks, inflammatory cells adhered to the endothelium and increased labeling of collagen was observed in tunica intima at both lesion-prone and non-lesion-prone sites, indicating endothelium activation. In plaques, internalized inflammatory cell density increased with age and plaque progression in tunicae adventitia and intima, but not media. In the whole plaque, aging or plaque progression did not alter the direct relationship between inflammatory cells and collagen. However, within the fibrous caps specifically, direct contact between inflammatory cells and collagen increased with age. This study demonstrates the potential of TPLSM in determining detailed information regarding the complex relationship between inflammatory cells and collagen during atherogenesis.

Imaging collagen in intact viable healthy and atherosclerotic arteries using fluorescently labeled CNA35 and two-photon laser scanning microscopy.

We evaluated CNA35 as a collagen marker in healthy and atherosclerotic arteries of mice after both ex vivo and in vivo administration and as a molecular imaging agent for the detection of atherosclerosis. CNA35 conjugated with fluorescent Oregon Green 488 (CNA35/OG488) was administered ex vivo to mounted viable muscular (uterine), elastic (carotid), and atherosclerotic (carotid) arteries and fresh arterial rings. Two-photon microscopy was used for imaging. CNA35/OG488 labeling in healthy elastic arteries was compared with collagen type I, III, and IV antibody labeling in histologic sections. For in vivo labeling experiments, CNA35/OG488 was injected intravenously in C57BL6/J and apolipoprotein E(-/-) mice. Ex vivo CNA35/OG488 strongly labeled collagen in the tunica adventitia, media, and intima of muscular arteries. In healthy elastic arteries, tunica adventitia was strongly labeled, but labeling in tunica media and intima was prevented by endothelium and elastic laminae. Histology confirmed the affinity of CNA35 for type I, III, and IV collagen in arteries. Strong CNA35/OG488 labeling was found in atherosclerotic plaques. In vivo applied CNA35/OG488 minimally labeled the tunica intima of healthy carotid arteries. Atherosclerotic plaques in apolipoprotein E(-/-) mice exhibited large uptake. CNA35/OG488 imaging in organs revealed endothelium as a limiting barrier for in vivo uptake. CNA35/OG488 is a good molecular imaging agent for atherosclerosis.

Both ADP and thrombin regulate arteriolar thrombus stabilization and embolization, but are not involved in initial hemostasis as induced by micropuncture.

OBJECTIVE: Thrombosis and embolization are main causes of morbidity and mortality. Up to now, the relative importance of mediators involved is only partly known. It was the aim of this study to investigate the involvement of ADP and thrombin in subsequent phases of arteriolar hemostasis and thromboembolism in vivo. METHODS: Rabbit mesenteric arterioles were punctured, which induced bleeding, hemostasis, and subsequent thromboembolism. This reaction as well as the activation state of platelets involved ([Ca(2+)](i)), was monitored in real time by intravital (fluorescence) microscopy. RESULTS: Neither inhibition of thrombin formation or thrombin activity nor blockade of platelet ADP receptors P2Y(1) and P2Y(12) influenced the initial hemostatic reaction: in all experiments initial bleeding was stopped by a primary thrombus within 2-3 s. On the other hand, both thrombin inhibition and P2Y(1) blockade increased rebleeding frequency, which indicates reduced thrombus stability in the long term. Finally, inhibition of either thrombin or ADP (via both receptors) reduced aggregate formation during the embolization phase by at least 90%. While most participating platelets exhibited a transient increase in [Ca(2+)](i) during embolization, an increased percentage of platelets showed no calcium response at all during P2Y(1) blockade, which was accompanied by reduced platelet-platelet interaction strength. CONCLUSIONS: Whereas thrombin and ADP are not involved in the initial hemostatic reaction, both substances appear to be essential to prevent rebleedings in the long term. During subsequent embolization, ADP (via both receptors) and small amounts of thrombin are involved in platelet activation.

Two-photon lifetime imaging of fluorescent probes in intact blood vessels: a window to sub-cellular structural information and binding status.

Fluorescence lifetime imaging (FLIM) provides a complementary contrast mechanism to fluorescence intensity and ratio imaging in intact tissue. With FLIM the time-resolved decay in fluorescence intensity of (interacting) fluorophores can be quantified by means of time correlated single photon counting (TCSPC). Here we focus on fluorescence lifetime imaging in intact blood vessels. Requisites for imaging in intact tissue are good penetration depth and limited tissue damage. Therefore, in this pilot-study, we performed TCSPC-FLIM using two-photon laser scanning microscopy to determine, with sub-cellular resolution, the fluorescence lifetime of two fluorescent probes. First, we focused on the nucleic acid dye SYTO41 in the various compartments of cells in vitro and in situ in the wall of intact mouse carotid arteries. Second, it was assessed whether the interaction of the lectin WGA-FITC with the endothelial glycocalyx affects its fluorescence lifetime. Results showed comparable mono-exponential fluorescence lifetimes of SYTO41 in the nuclei of cells in vitro and in situ. The slightly shorter fluorescence lifetime observed in the cytoplasm allowed discrimination of the nuclei. SYTO41 displayed strong mitochondrial staining, as was verified by the mitochondrion-specific probe CMXRos. In addition, mitochondrial staining by SYTO41 was accompanied by a green shift in emission. In the mitochondrial region, SYTO41 showed a highly bi-exponential and relatively fast decay, with two distinct lifetime components. It is hypothesized that the fitted bi-exponential decay can either be contributed to (1) the mathematical approximation of the fluorescence intensity decay or (2) the presence of free and DNA-bound SYTO41 in the mitochondrial compartment, leading to two lifetime components. The fluorescence lifetime of WGA-FITC decreased by approximately 25% upon binding to the endothelial glycocalyx. From this study, we conclude that FLIM offers an additional contrast mechanism in imaging intact tissue and provides information on binding status between a probe and its ligand.

Optical and magnetic resonance imaging of cell death and platelet activation using annexin a5-functionalized quantum dots.

A quantum-dot-based nanoparticle is presented, allowing visualization of cell death and activated platelets with fluorescence imaging and MRI. The particle exhibits intense fluorescence and a large MR relaxivity (r1) of 3000-4500 mM-1 s-1 per nanoparticle due to a newly designed construct increasing the gadolinium-DTPA load. The nanoparticle is suitable for both anatomic and subcellular imaging of structures in the vessel wall and is a promising bimodal contrast agent for future in vivo imaging studies.

The endothelial glycocalyx: composition, functions, and visualization.

This review aims at presenting state-of-the-art knowledge on the composition and functions of the endothelial glycocalyx. The endothelial glycocalyx is a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Both endothelium- and plasma-derived soluble molecules integrate into this mesh. Over the past decade, insight has been gained into the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cell-vessel wall interactions. The contribution of the glycocalyx to diabetes, ischemia/reperfusion, and atherosclerosis is also reviewed. Experimental data from the micro- and macrocirculation alludes at a vasculoprotective role for the glycocalyx. Assessing this possible role of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. An overview is given of the various ways in which the endothelial glycocalyx has been visualized up to now, including first data from two-photon microscopic imaging.

Preservation of rat cremaster muscle microcirculation after prolonged cold storage and transplantation.

BACKGROUND: Microvascular surgery for the reconstruction of complex defects involves an ischemic period, which may cause flap failure as the result of ischemia/reperfusion injury. We assessed the microvascular consequences of rat cremaster muscle transplantation after prolonged periods of cold storage in HTK-Bretschneider solution (HTK). MATERIALS AND METHODS: Cremaster muscle transplantations were performed immediately or after 8 or 24 h of cold storage (4 degrees C) in HTK or saline. Intravital microscopy was used to quantify capillary perfusion and venular leukocyte-endothelium interactions following transplantation. RESULTS: The transplantation procedure itself resulted in 50-65 min of ischemia. After direct transplantation, capillary perfusion was 90% of control. Transplantation after 8 h of cold storage in either HTK or saline did not deteriorate capillary perfusion. When the tissue was stored for 24 h, HTK was superior to saline in preserving capillary perfusion (HTK: 76-83% of control, saline: 30%). Immediate transplantation induced a small increase in leukocyte adhesion. Prolonged cold storage in either fluid resulted in reduced flow velocities (qualitative observations) and edema formation, which hampered quantification of leukocyte-endothelium interactions. CONCLUSIONS: Even after 8 or 24 h of cold storage in HTK, transplantation of rat cremaster muscle was successful with good capillary perfusion. Capillary perfusion was better preserved in HTK than in saline.

Regulation of microvascular thromboembolism in vivo.

Atherothrombosis and embolization are main causes of morbidity and mortality in the Western world. To optimize treatment, better understanding of the factors involved in thromboembolism in vivo is needed. The course and outcome of a thromboembolic process are determined by the local balance between anti and prothrombotic factors. In healthy vessels, endothelial antithrombotic properties prevent blood platelets from interacting with the vessel wall. Upon vessel wall damage or endothelial activation, however, prothrombotic factors temporarily overrule the antithrombotic factors, leading to thrombus formation and embolization. According to this concept, thromboembolism ends when the balance is restored. Animal models on microvascular thromboembolism have provided evidence that the endothelium is eminently involved in the regulation of thromboembolism, and that shear forces are an important determinant of endothelial function. Therefore, in this review focus is on the endothelial regulation of platelet-vessel wall interactions during thromboembolism in vivo. Anti- and prothrombotic properties of vascular endothelium will be discussed, paying special attention to the endothelium-derived platelet inhibiting substances nitiric oxide (NO) and prostacyclin (PGl(2)) and to differences between arteriolar and venular endothelium. In addition, the involvement of shear forces in microvascular thromboembolic processes in vivo will be described

Do preservation solutions protect rat cremaster microcirculation during ischemia and reperfusion?

BACKGROUND: Our aim was to investigate the potential of the preservation solution Celsior to protect rat cremaster muscle microcirculation during ischemia and reperfusion, and to compare its effects with those of HTK (histidine-tryptophan-ketoglutarate-Bretschneider solution). Because of its anti-oxidant contents, we expected Celsior to be more protective than HTK. MATERIALS AND METHODS: Capillary perfusion and leukocyte-endothelium interactions were examined in rat cremaster muscle using intravital microscopy. After perfusion with Celsior or HTK (4 degrees C), the cremaster was subjected to 4 or 6 h of warm (33-34 degrees C) ischemia and 2 h of reperfusion. Measurements were performed prior to perfusion and/or ischemia, and 0, 1, and 2 h after restoration of flow. RESULTS: Without Celsior or HTK, capillary perfusion transiently decreased to 50% of baseline after 4 h of ischemia; it remained low (45%) after 6 h of ischemia. Whereas HTK had no significant influence, Celsior deteriorated capillary perfusion: it remained low after 4 h of ischemia (39-48%) and decreased even further after 6 h of ischemia (18-8%). Both preservation solutions similarly reduced the increase in leukocyte-endothelium interactions after ischemia. CONCLUSIONS: Preischemic tissue perfusion with Celsior had an adverse effect on capillary perfusion in rat cremaster muscle after 4 and 6 h of ischemia, whereas HTK did not significantly influence this parameter. Both preservation solutions similarly prevented the increase in leukocyte-endothelium interactions after ischemia. These data suggest that HTK is more suited as a preservation solution for muscular tissue than Celsior, especially when the known protective effects of HTK on muscle function are taken into account.

Effect of HTK on the microcirculation in the rat cremaster muscle during warm ischemia and reperfusion.

Histidine-tryptophan-ketoglutarate (HTK) preserves rat muscle function during cold storage. We examined the effect of HTK perfusion on preservation of microvascular function during 4 h of warm ischemia and subsequent reperfusion (I/R) in the rat cremaster muscle. Leukocyte-endothelium interactions, capillary perfusion, and arteriole diameters were quantified prior to HTK-perfusion and/or ischemia, and at 0, 1, and 2 h after restoration of blood flow. In all groups, the number of rolling leukocytes increased with time, whereas I/R induced a slight increase in leukocyte adhesion. After ischemia, capillary perfusion rapidly recovered to about 50% and returned to near normal (90%) after 2 h. HTK at 22 degrees C did not affect the assessed microcirculation variables, whereas HTK at 4 degrees C reduced leukocyte rolling, but not adhesion. Therefore, microvascular function of HTK-perfused muscles was not better preserved during warm I/R than that of nonperfused muscles. Contrary to other preservation solutions, HTK perfusion in itself was not detrimental to the microcirculation.

Effect of hypothermia and HTK on the microcirculation in the rat cremaster muscle after ischaemia.

Hypothermia is an important preservation method for tissues and solid organs. The aim of the present study was to assess in rat cremaster muscle the effect of hypothermia, without or with pre-ischaemic HTK (histidine-tryptophan-ketoglutarate-Bretschneider solution) perfusion, on microvascular consequences of 4 or 6 h ischaemia and 2 h of reperfusion. Intravital microscopy was applied to examine capillary perfusion and leucocyte-endothelium interactions. The cremaster muscle was subjected to 4 or 6 h of cold (4 degrees C) or warm (33-34 degrees C) ischaemia and 2 h of reperfusion. Measurements were performed at baseline, prior to HTK perfusion and ischaemia, and at 0, 1 and 2 h after blood flow restoration. Hypothermia completely prevented the 50% reduction in capillary perfusion that was observed previously at start of reperfusion after 4 h warm ischaemia. After 6 h of warm ischaemia, perfusion resumed in only 45% of capillaries and remained at this low level during reperfusion. In contrast, only a slight decrease (< 10%) in capillary perfusion was observed after 6 h of cold ischaemia. Pre-ischaemic HTK perfusion had no beneficial effect on tissue perfusion. Both hypothermia and HTK attenuated the significant increase in venular leucocyte-vessel wall interactions, which was observed after 4 h of warm ischaemia in a previous study. Combined application of both interventions had no additional effects. After 6 h of warm ischaemia, no increase in leucocyte-vessel wall interactions was observed, possibly due to venular flow reduction. In conclusion, hypothermia preserves capillary perfusion and prevents an increase in leucocyte-vessel wall interactions during reperfusion after muscle tissue ischaemia. Preischaemic perfusion of the vasculature with HTK does not improve the effects of cold storage on tissue perfusion, but attenuates the inflammatory response independently of temperature effect.

Two-photon microscopy for imaging of the (atherosclerotic) vascular wall: a proof of concept study.

BACKGROUND: Understanding atherogenesis will benefit significantly from simultaneous imaging, both ex vivo and in vivo, of structural and functional information at the (sub)cellular level within intact arteries. Due to limited penetration depth and loss of resolution with depth, intravital and confocal fluorescence microscopy are not suitable to study (sub)cellular details in arteries with wall thicknesses above 50 microm. METHODS: Using two-photon laser scanning microscopy (TPLSM), which combines 3D resolution and large penetration depth, we imaged mouse carotid arteries. RESULTS: In thin slices, (sub)cellular structures identified using histochemical techniques could also be identified using TPLSM. Ex vivo, structural experiments on intact atherosclerotic arteries of Apo-E(-/-) mice demonstrated that in contrast to confocal or wide-field microscopy, TPLSM can be used to visualize (sub) cellular structural details of atherosclerotic plaques. In vivo, pilot experiments were carried out on healthy arteries of wild-type C57BL6 and atherosclerotic arteries of Apo-E(-/-) mice. As an example of functional measurements, we visualized fluorescently labeled leukocytes in vivo in the lumen. Additionally, detailed morphological information of vessel wall and atherosclerotic plaque was obtained after topical staining. CONCLUSIONS: Thus, TPLSM potentially allows combined functional and structural studies and can therefore be eminently suitable for investigating structure-function relationships at the cellular level in atherogenesis in the mouse.

Gas plasma treatment: a new approach to surgery?

In this survey we analyse the status quo of gas plasma applications in medical sciences. Plasma is a partly ionized gas, which contains free charge carriers (electrons and ions), active radicals, and excited molecules. So-called nonthermal plasmas are particularly interesting, because they operate at relatively low temperatures and do not inflict thermal damage to nearby objects. In the past two decades nonthermal plasmas have made a revolutionary appearance in solid state processing technology. The recent trends focus on using plasmas in health care, for "processing" of medical equipment and even living tissues. The major goal of tissue treatment with plasmas is nondestructive surgery: controlled, high-precision removal of diseased sections with minimum damage to the organism. Furthermore, plasmas allow fast and efficient bacterial inactivation, which makes them suitable for sterilization of surgical tools and local disinfection of tissues. Much research effort must be undertaken before these techniques will become common in medicine, but it is expected that a novel approach to surgery will emerge from plasma science.

In vivo blockade of platelet ADP receptor P2Y12 reduces embolus and thrombus formation but not thrombus stability.

OBJECTIVE: ADP is a key platelet agonist in thromboembolism. One of the receptors involved in ADP-induced platelet activation is the P2Y12 receptor, which is a target for antithrombotic drugs. METHODS AND RESULTS: Here, we present first evidence for a differential role of this receptor in thrombus and embolus formation in vivo. Anesthetized rabbits were treated with the selective P2Y12 antagonists AR-C69931 MX (3 microg x kg x min(-1) IV) or clopidogrel (25 mg/kg orally). Efficacy of these treatments was monitored by aggregation and thrombin generation measurements in blood samples ex vivo. Mesenteric arterioles were mechanically injured; thrombus growth and subsequent embolus formation were visualized by real-time intravital microscopy. AR-C69931 MX and clopidogrel significantly (P<0.05) reduced the total duration of embolization (by 52% and 36%, respectively), and fewer and smaller emboli were produced. The size of the initial thrombus was significantly reduced (P<0.005), but its stability was unaffected: plug formation was still effective. CONCLUSIONS: These findings demonstrate that ADP and its P2Y12 receptor are involved in thrombus growth and especially in the formation of emboli on the downstream side of the initial thrombus. This may explain the beneficial effects of P2Y12 receptor antagonists in secondary prevention of ischemic events in patients with arterial thrombosis.

In vivo monitoring of intracellular free calcium changes during uterine activation by prostaglandin f(2alpha) and oxytocin.

OBJECTIVE: It has been well established that oxytocin (OXT) increases intracellular free calcium ([Ca(2+)](i)) by targeting both intracellular and extracellular stores, but the mechanisms involved in the increase through activation with prostaglandin F(2alpha) (PGF(2alpha)) are still incompletely understood. This study was designed to elucidate the source(s) of increased [Ca(2+)](i) in response to PGF(2alpha) (10(-6) M) or OXT (10(-8) M) administration in the near-term rat myometrium. METHODS: The animals were divided into an in vitro group (n= 8), where the developed tension of uterine strips was assessed, and an in vivo group (n= 5), where a lobe of the uterus with intact innervation and circulation was loaded with the fluorescent indicator Indo-1 AM to assess [Ca(2+)](i). RESULTS: PGF(2alpha) and OXT induced a 30.1% and 35.9%, respectively, increase in developed tension in the potassium chloride-depolarized myometrial strips. Nifedipine reduced the PGF(2alpha) and OXT increased tension by 65.8% and 49.4%, respectively. In vivo, both PGF(2alpha) and OXT increased [Ca(2+)](i) in the potassium chloride-depolarized uterine muscle by 35.7% and 44.6%, respectively, increases similar to the rises in tension in vitro. Nifedipine reduced these effects of PGF(2alpha) and OXT by 45.3% and 39.6%. CONCLUSION: These findings indicate that in near-term myometrium the source of increased [Ca(2+)](i) after administration of PGF(2alpha), similar to OXT, is both extracellular and intracellular.