A 3D reconstruction of pancreas development in the human embryos during embryonic period (Carnegie stages 15-23).

AIMS: The goal in this paper was to rebuild a three dimensional (3D) reconstruction of the dorsal and ventral pancreatic buds, in the human embryos, at Carnegie stages 15-23. METHOD: The early development of the pancreas is studied by tissue observation and reconstruction by a computer-assisted method, using a light micrograph images from consecutive serial sagittal sections (diameter 7 microm) of ten human embryos ranging from Carnegie stages 15-23, CRL 7-27 mm, fixed, dehydrated and embedded in paraffin, were stained alternately with haematoxylin-eosin or Heindenhain'Azan. The images were digitalized by Canon Camera 350 EOS D. The serial views were aligned automatically by software, manual alignment was performed, the data were analysed following segmentation and threshold. RESULTS: The two buds were clearly identified at stage 15. In stage 16, both pancreatic buds were in final position, and begin to merge in stage 17. From stage 18 to the stage 23, surrounding connective tissue differentiated. In the stage 23, the morphology of the pancreas was definitive. The superior portion of the anterior face of the pancreas's head was arising from the dorsal bud. The rest of the head including the uncinate process emanated from the ventral bud. CONCLUSION: The 3D computer-assisted reconstruction of the human pancreas visualized the relationships between the two pancreatic buds. This explains the disposition and the modality of the components fusion. This embryologic development permits a better understanding of congenital abnormalities.

Differential expression of voltage-gated Ca(2+)-currents in cultivated aortic myocytes.

The expression of different types of Ca(2+)-channels was studied using the whole-cell patch-clamp technique in cultured rat aortic smooth-muscle myocytes. Ca(2+)-currents were identified as either low- or high voltage-activated (ICa,LVA or ICa,HVA, respectively) based on their distinct voltage-dependences of activation and inactivation, decay kinetics using Ba2+ as the charge carrier and sensitivity to dihydropyridines. The heterogeneity in the functional expression of the two types of Ca(2+)-channels in the cultured myocytes delineated four distinct phenotypes; (i), cells exhibiting only LVA currents; (ii), cells exhibiting only HVA currents; (iii), cells exhibiting both LVA and HVA currents and (iv), cells exhibiting no current. The myocytes exclusively expressed HVA currents both during the first five days in primary culture and after the cells had reached confluence (> 15 days). In contrast, LVA currents were expressed transiently between 5 and 15 days, during which time the cells were proliferating and had transient loss of contractility. Thus, both LVA and HVA Ca(2+)-current types contribute to Ca(2+)-signalling in cultured rat aortic myocytes. However, the differential expression of the two Ca2+ current types associated with differences in contractile and proliferative phenotypes suggest that they serve distinct cellular functions. Our results are consistent with the idea that LVA current expression is important for cell proliferation.

Decrease of bradykinin-induced glomerular contraction in diabetic rat: a new cellular interpretation.

The contractile response to bradykinin (BK), measured by the reduction of the planar surface area, was studied in glomeruli and mesangial cells (MC) isolated from diabetic rats (D) one week after diabetes induction with injection of streptozotocin (STZ; 60 mg kg-1, i.p.). Results were compared with age and weight-matched untreated rats (N) and were expressed by two parameters of cell activity, the mean maximum contraction (MMC) and the proportion of contractile cells (PCC). Glomerular and mesangial contraction were found to be clearly reduced in diabetic rats in response to 100 nM BK. The lower contractile response was associated with a decrease of both glomerular calcium uptake and mesangial cell intracellular calcium mobilization. The fact that cell pretreatment with two protein kinase C (PKC) inhibitors, phorbol 12-13 myristate acetate and calphostin, lowered normal cell contraction at the level of that found in diabetic MC without any significant effect in the latter, suggests the involvement of a PKC pathway, perhaps by a decrease of activatable PKC in diabetes. In addition, our results led to the first description of a possible role of the kallikrein-kinin system in the early glomerular hemodynamic changes occurring in diabetes. Insulin (1-200 nM) increased the contractile response of cultured diabetic cells (MMC), and in this case, it also increased the PCC. It must be stressed that the effect of 1 nM insulin on the former (88% increase) was very much smaller than its effect on the latter (103% increase). The combination of the two parameters (contraction index, CI) provided a realistic evaluation of the contractile capacities of the cell population of the cultures as a whole. The differences in this index between normal and diabetic cell populations, in the absence or presence of insulin, were strictly parallel to those found in intact glomeruli. Finally, our results further confirm (Ouardani et al., Biol. Cell 86, 127, (1996)) the limit of the first five cell passages within which cultured MC can be reasonably used for the study of contractile abnormalities occurring in the early steps of diabetic state.

Activation of phosphatidylinositol synthesis by different agonists in a primary culture of smooth muscle cells grown on collagen microcarriers.

Regulation of inositol phosphate synthesis was examined in a primary culture of vascular smooth muscle cells grown on collagen-coated microcarriers. In the presence of LiCl (10 mM), four agonists [serotonin, angiotensin, (arginine) vasopressin and noradrenaline] were found to stimulate the formation of inositol phosphates in a dose-dependent manner. All agonists were found to have identical and additive effects on the time course of inositol phosphate formation. Therefore, our primary cell culture technique was proved to give smooth muscle cells suitable for the study of modulation of phosphoinositide metabolism in response to physiological effectors.

Maitotoxin stimulates the formation of inositol phosphates in rat aortic myocytes.

Maitotoxin is the most potent of the known marine toxins. The effect of maitotoxin on muscle contraction or hormone release was consistent with its action on the voltage-sensitive channel. Indeed, calcium antagonists such as nifedipine or diltiazem were able to reverse the maitotoxin effects. Using smooth muscle cells, we have analysed the effects of maitotoxin on the inositol phosphate metabolism. Maitotoxin stimulates the inositol phosphate formation (5 +/- 1.8-fold in the presence of 10 mM LiCl). Moreover, this effect is not reversed, even partially by calcium antagonists, by alpha 1 antagonists and is not mimicked by Ca2+ ionophores such as A23187 or calcium agonists such as Bay-K 8644. The action of maitotoxin is further discussed in this paper.

Modulation of the accumulation of inositol phosphates and the mobilization of calcium in aortic myocytes.

In vascular smooth muscle cells the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA), a potent activator of C-kinase, inhibited the accumulation of inositol phosphates and the mobilization of calcium produced by several agonists. In the same way, TPA inhibited the fluoride-induced activation of phosphoinositide metabolism. These results suggest a C-kinase action at a post-receptor level. Moreover, the fluoride-induced accumulation of inositol phosphates shows the presence of one or more guanine nucleotide-binding proteins (G-proteins) in the regulation of receptor-phospholipase C coupling. This was confirmed by the use of N-ethylmaleimide and pertussis toxin. These results support the view that, in addition to the induction of sustained contractions, C-kinase can activate negative feedback mechanisms in aortic myocytes.

Contribution to the 3D computer assisted reconstruction of pancreatic buds in the rat embryos.

The aim of this work was to reconstruct, in the rat embryos, stage 12-23, the three dimensional (3D) distribution of the dorsal and ventral pancreatic buds by of a computer assisted method. Ninety-six rat embryos, CRL 3-16 mm, fixed, dehydrated, and paraffin embedded, were submitted to serial histological sections and stained by hematoxylin-eosin and Heidenhain's azan techniques. The images were digitalized by Canon Camera 350 EOS D. The serial views were aligned anatomically by software and the data were analyzed following segmentation and thresholding. The dorsal pancreas developed from the dorsal wall of the duodenum in stage 12, while the ventral pancreas arose from the ventral wall of the hepatic diverticulum in stage 13 and 14. The rotation of ventral pancreas started in stage 15 and was completed in stage 16. The fusion of both buds was evident in stage 17. In stage 23 the limit between dorsal and ventral bud was still marked by the pathway of superior mesenteric vein.

Discrimination between human melanoma cell lines by fluorescence anisotropy.

The fluorescence polarization of diphenylhexatriene (DPH) and trimethylammonium diphenylhexatriene (TMA-DPH) was measured when these markers were imbedded in cells of the human melanoma cell lines IGR37, IGR39, IGR3 and IGR4, as well as in cells of the mouse melanoma cell lines B16F1 and B16 F10. These measurements were performed on cell cultures which were grown on quartz plates as well as on cell suspensions. Considerable differences are found between the polarization values of the human cell lines that are related to their different origins. Differences for the plated cells are considerably greater than those for the suspensions. No differences in the polarization values were found for the two mouse melanoma lines. It is concluded that differences in lipid structural order can be found between cell types endowed with different metastasizing capabilities.

Calf-spleen nicotinamide--adenine dinucleotide glycohydrolase. Solubilization purification and properties of the enzyme.

NAD glycohydrolase of calf spleen was solubilized with pancreatic lipase and purified approximatively 800-fold to a specific activity of 7 units/mg of protein by successive DEAE-cellulose and carboxymethyl-cellulose chromatography. The purified enzyme has a molecular weight of 24,000 and is characterized by a double band on disc gel electrophoresis. Some kinetic properties of the NAD-glycohydrolase-catalyzed hydrolsis of NAD have been examined using a titrimetric assay for enzyme activity. The reaction is subject to inhibition be excess of substrate, which disappears at high ionic strength and low pH. At a pH below 5 the kinetic displays an apparent activation by substrate. The effects of pH (4.5-9.0) on the kinetic parameters do not reveal an essential ionizable group in the catalytic process.

Effects of naftidrofuryl on the contraction and proliferation of cultured myocytes evoked by serotonin.

We investigated the effects of 2-(diethylamino)-ethyl-tetrahydro-alpha-(l-naphthylmethyl)-2-furanpro pionate (naftidrofuryl) on both the contraction and the proliferation of cultured rat aortic myocytes elicited by serotonin. Cells were cultured under controlled conditions, both on microcarrier beads and in microwells. The results show that naftidrofuryl alone had no effect on the properties of vascular smooth muscle cells. However, in the presence of serotonin, naftidrofuryl inhibits the contraction and the proliferation of smooth muscle cells.

Calf spleen NAD glycohydrolase. Comparison of the catalytic properties of the membrane-bound and the hydrosoluble forms of the enzyme.

The catalytic properties of membrane-bound calf spleen NAD glycohydrolase were studied in comparison with previous data obtained with a solubilized hydrosoluble form of the enzyme. When the hydrolysis of NAD catalyzed by membrane-bound NAD glycohydrolase was studied at pH values below 7.5, only insignificant interference by other NAD-hydrolyzing enzymes was detected, and no proton-diffusional inhibition was observed. The kinetics could, therefore, be followed using a titrimetric assay for NAD glycohydrolase activity. The effect of pH, ionic strength on the kinetic parameters, and shifts in binding constants for several ligands of the membrane-bound enzyme indicate that the NAD glycohydrolase activity is influenced by an electrostatic potential due to negative charges (polyelectrolyte effect). No significant changes in kinetic mechanism could be found between both NAD glycohydrolase forms. The association of the enzyme with the membrane results in a remarkably increased thermal stability, in changes in binding properties of the active site and in the emergence of new inhibitor binding sites; e.g. adenosine 3':5'-monophosphate (cyclic AMP) and adenosine, which do not inhibit the hydrosoluble form of NAD glycohydrolase, are good inhibitors (respectively competitive and mixed) of the membrane-bound enzyme. These data (i.e. allotopic changes) probably can be ascribed to enzyme conformational changes induced and stabilized by interaction with membrane constituents.

Differences in proliferation of primary cultures of vascular smooth muscle cells taken from male and female rats.

Vascular smooth muscle cells from thoracic aortas of 10-week-old rats were cultured on different substrates. Cells taken from male animals proliferated more quickly than those taken from females; this was true for all substrates, but the difference was accentuated by growth on collagen membranes. The relationship between these results and the sex-linked incidence of hypertension and atherosclerosis is discussed and a model for future studies proposed.

An hypothesis for the interpretation of the contractile response of vascular smooth muscle at the cellular level.

The long preservation and recovery of functional (contractile) properties in cultured aortic smooth muscle cells, even after replating or deep-frozen storage and the measurement of their responses are now technically settled issues. We could thus study extensively the responses of single cultured cells from rat thoracic aorta. Responses were elicited by the addition of KCl 40 mmol/L without or with a calcium blocker PN 200-100 (10(-6) mol/L); angiotensin II (10(-11)-10(-6) mol/L) without or with antagonist (losartan 10(-5) mol/L); or serotonin (10(-9)-10(-4) mol/L) without or with antagonist (naftidrofuryl 10(-5) mol/L). Results thus obtained enabled us to propose a new hypothesis for the interpretation of the contractile responses of an elastic vascular smooth muscle. The different maximal effects of different agonists result mainly from the different proportions of cells they can mobilize; the agonist concentration-contraction relationship is mainly due to the increase of the proportion of cells involved up to a maximal value typical of the agonist used. An antagonist primarily reduce the proportion of cells an agonist can mobilize. Some of the consequences of this hypothesis are briefly outlined.

Calf-spleen nicotinamide-adenine dinucleotide glycohydrolase. Properties of the active site.

The interaction between the nicotinamide adenine dinucleotide binding domain of calf spleen NAD glycohydrolase and its ligands has been studied. The use of competitive inhibitors, structurally related to different portions of the NAD molecule (i.e. adenosine and nicotinamide moieties), revealed the considerable importance of the binding between the pyrophosphate linkage and probably an arginyl residue of the active site. This interaction allows the positioning of the substrate in a conformation which permits catalysis to occur. The binding between the 2'-hydroxyl of the adenosine moiety and a residue of the active site, which exists in NAD-linked dehydrogenases, is probably missing in the calf spleen NAD glycohydrolase, based on the inhibition by salicylates, 2'-deoxyadenosine 5'-monophosphate and the hydrolysis of the 2'-deoxyadenosine analogue of NAD. The NAD glycohydrolase could be completely inactivated by 2,3-butanedione, an arginyl-modifying reagent. The reaction followed pseudo-first-order kinetics and the modification was found to be reversible. Woodward's reagent K, a reagent for carboxyl residues, partially inactivated the enzyme, which resulted in a change of the NAD glycohydrolase kinetic parameters Km and V. The inactivation rate was complicated by a parallel decomposition of the reagent.

Differences in myocyte subpopulations from segments of the thoracic aorta and their modifications with age and hypertension in the rat.

Smooth muscle cells obtained from three distinct segments of the thoracic aorta of both Wistar Kyoto (WKY) and Spontaneously Hypertensive Rats (SHR) taken at different stages of development were studied in terms of their volume, DNA content in single cell suspensions, and doubling time in primary cultures. The proliferative activity and mean cell volume of myocytes from WKY rats increase along the thoracic aorta in a gradient from the aortic arch to the diaphragm. The slope of this gradient is increased in SHR because of an extension of the area that contains myocytes with low proliferative activity in primary cell culture and large cell volumes in suspension. Tetraploid myocytes are found in both strains and their proportions are larger in SHR than in WKY, specifically after the onset of hypertension. However, they appear to be evenly distributed along the thoracic aorta with a size distribution that is included in that of the diploid cells from the same area. It is suggested that changes in the structural properties of the aortic-cell compartment, associated with maturation and hypertension, reflect quantitative changes in the relative proportions of several myocyte subpopulations within the aorta of the rat.

Quantitative method for the study of the morphological changes induced by vasoactive agents in single aortic myocytes grown in primary cultures.

Morphological changes of aortic myocytes grown in primary cultures on collagen gels, were induced by two adrenergic agonists, in the absence or presence of specific antagonists. Their effects were quantified by means of the measurement of the surface areas of the images of the cells. The alpha-adrenoreceptor agonist phenylephrine induced a concentration-dependent decrease of the surface areas of the images of the cells, which was competitively antagonized by phentolamine, and was reduced in the presence of CdCl2 (an inhibitor of calcium entry). The beta-adrenoreceptor agonist isoproterenol induced an increase in the surface areas of the images of prostaglandin F2-alpha pretreated cells and this effect was antagonized by propranolol. It is concluded that the morphological changes of myocytes in primary culture, induced by phenylephrine or isoproterenol can be quantified by the measurement of the surface areas of their images. These changes were in good agreement with the response of isolated thoracic aorta rings or strips to the same agonists with regard to their sensitivity, specificity, and kinetic features. Thus the measurement of the changes of the surface area of the images of myocytes induced by vasoactive agents constitutes a noninvasive, nondestructive method for the quantification of their responses to these agents at the single cell level.

Study of living single cells in culture: automated recognition of cell behavior.

An automated system capable of analyzing the behavior, in real time, of single living cells in culture, in a noninvasive and nondestructive way, has been developed. A large number of cell positions in single culture dishes were recorded using a computer controlled, robotized microscope. During subsequent observations, binary images obtained from video image analysis of the microscope visual field allowed the identification of the recorded cells. These cells could be revisited automatically every few minutes. Long-term studies of the behavior of cells make possible the analysis of cellular locomotary and mitotic activities as well as determination of cell shape (chosen from a defined library) for several hours or days in a fully automated way with observations spaced up to 30 minutes. Short-term studies of the behavior of cells permit the study, in a semiautomatic way, of acute effects of drugs (5 to 15 minutes) on changes of surface area and length of cells.

Smooth muscle cell hypertrophy and hyperplasia in the thoracic aorta of spontaneously hypertensive rats.

Changes in the smooth muscle cell compartment (SMCC) of the media layer of the aorta were studied in spontaneously hypertensive (SHR) and in normotensive rats (WKY) of both sexes between 3 to 18 weeks of age. Up to 7 weeks of age, development of the SMCC occurred in males and females of the two strains both through a massive increase in cell number and in cell size. In SHR after 7 weeks of age, the development of the SMCC was due to a marked increase in cell size together with an increase in cell number. In contrast during the same period, the development of the SMCC in WKY was associated almost exclusively with an increase in cell size. It is concluded that the presence of a greater number of larger smooth muscle cells contributes to the hypertrophy of the arterial wall of hypertensive animals.

[Expression of results in studies of vascular biochemical components during the development of the SHR rat]. / Problèmes liés à l'expression des résultats dans les études des composants biochimiques des vaisseaux durant le développement du rat hypertendu SHR.

Hypertrophy and hyperplasia induced by age and hypertension as well as tissue heterogeneity makes it difficult to analyse biochemical datas obtained with rats thoracic aortas from rats of various age and blood pressure. In SHR, the thoracic aorta weight increases parallely to the body weight from the first week after birth onwards. Meanwhile the protein content of the organ increases parallely to the organ weight. However, though the DNA content increases markedly up to 7 weeks of age, one observes a much lower increase with age afterwards. It seemed to us that the aorta DNA content was more representative of the organ cell number. This led us to use express our biochemical data per mg of the organ DNA content. Moreover, it seems necessary to take in account the cell volume which increases as well with age and hypertension on the one hand, and to be able to distinguish the cellular content of the myocytes from the one of other cells types contained in the adventitia and the intima, on the other hand. For these purposes pure media layers are prepared by completely removing the adventitia and intima and single cell suspensions are obtained after total enzyme digestion of the medias.

Mechanical responses of rat vascular smooth muscle cells to rat interferon.

Mechanical responses elicited by rat interferon (IFN) were investigated in rat vascular smooth muscle cells (RVSMC) in primary culture. IFN induced a dose-dependent reduction in the apparent cell surface area, which was quantitatively comparable to, or even greater than, the response elicited by the alpha-adrenergic agent, phenylephrine. Neither mock-IFN nor rat IFN-neutralized with specific antibodies elicited such a response. The sensitivity of RVSMC to the mechanical effect of IFN was species specific. In addition, the effect of IFN was inhibited by the Ca2+ entry blocker, verapamil.