P-C1-33 Purinergic receptors in smooth muscle cells isolated from rat small pulmonary arteries

C 1 Transmembrane signalling and transduction

Use of the patch-clamp recording technique to determine which P, urinoceptor subtypes are present on rat smalP pulmonary arteries(200-4OOpMin diameter) and to examine the consequencesof their stimulation. Methods:’ Single pulmonary arterial smooth muscle cells (PASMC’s) were isolated using an enzymatic dispersion ’ procedure and i&ic currents were measured in either the whole-cell or perforated- atch recording configuration. Results: ADD P‘cation of 1OuM ATP toPASMC’s maintained’;n the perforated-patch configuration resulted in the activation of a fast, transient nonselectiveinward current (I,,,) and an oscillatory inward Cr current (I,&. ‘I’he order of potency for agonist induced activation of I ,Ns was 2methylthio ATp> ATP> a,&methyiene ATP> ADP> UTP, whereas for Iclcl it was UTP= ATP> ADP> 2-methylthio ATP> cc,&methylene ATP. Activation of IC’,cawas dependent upon elevation of intracellular Ca” (Ca2’i) and was inhibited by buffering Ca2’i with EGTA in the whole-cell configuration (IT.NS was unaffected). Conclusions: We conclude, therefore that PASMC’s contain both P,, and P,, receptor subtypes. P,, receptor stimulation causes Ca2+ release from intracellular stores resulting in activation of I,,,,, while P,, receptor stimulation resultsin the activation of I,,,.

P-cl-34 RBCIJIATlON OF INCELLSAND FORDP, ToRtipNO R, M, PARIS1M. Dept Physiol, Fat Med, UBA, BuenosAires (ARG)and Dept Biol Ceil and Mol, CESaclay,(Fr) Porpoise: To test the role and hormonal regulation of aquaporins in mammalian cells and mammalian epithelial cell layers. Methods: The osmotic permeability (Posm)was measured, employing videomicroscopy, in ovary and oviduct rat oocytes. Posm was also measured in reconstituted epithelial barriers formed with the T84 cell line. The short circuit current (SCC)and the net water flux (Jw)were simultaneously measured. Structural changes were monitored by electron microscopy. Results: Rat oocytes Posm was higher in the proestro than in the estro. Sensitivity to mercurials was only observed in the proestro and could be reverted with mercaptoethanol. In T84 epithelial cell layers the SCC went progressively down after mounting, in parallel with a reduction in the observed se&etory Jw. A23187, a well know Cl- secretagoge, induced a transient increasein SCCwith no change in Jw. Conclusions: Aquaporins would be present in the cell membrane of the rat oocyte during the early stages of development and would disappear after ovulation. In T84 cell layers a transport associated Jw was observed, in secretory conditions. Nevertheless, after A23187 stimulation, a Jw/SCCuncoppling was observed.

P-Cl-35

P-Cl-36

BINDING OF IGF-I AND IGF-II TO TBEIR RECEPTORS AND BINDING PROTEINS IN RAT Cc GLIOMA CELLS.

SPONTANEOUS INTRACELLULAR FREE C LCIUbf SPHCESSNBVCED BY FLUID #L OW APPLICATION TO CULTURED ENDOTHELIAL CELLS

P-Cl-33 PURuy@I?GIC

MUWE-

RECEPTORS

SMALL4VLMONAIW

IN SMUOTH RAT AR’I’ERIES

i!WLATED FBDM

HARTLEY. S.A. & KCZLOWSKI. R.Z. UniversityDeptof F’hannacology, Oxford(UK) Purpose:

WANG Q., SLEGERSH., CLAUWAERT J. Biophysics Research Group, Department of Biochemistry, U.A.,Antwerp(Belgium) Purpose: The action of IGFs in the central nervous system is presumed to be mediated by biidii to specific receptors.We have used Cg s&ma cells as a model system for the study of &heIGFs , their receptors&d biding pro&s. Methods: We have used ‘2SI-labeledIGF oeotides to study ligand bindii to Cs ‘&l membranes, solubilii rerceptor fractions and IGFBPs. After high affinity binding, thi! labeled IGFs were chemically cross-linked to crude plasma membranes preparations, solubilized receptor fractions or serum-free conditioned medium, using disuccinimidyl suberate. Results and Conclusion: The IGF-I receptors have an association constant k. of 1.2x10 M’ for the IGF-I peptide and the IGF-II receptors bind the IGF-II peptide with a k, of 2.5x10 M’ The receptorsappear identical to reported IGF-I and IGF-II receptors in peripheral tissues and are presumable capable of mediating the biological actions of both IGF-I and IGF-II. Similarly, Cb secretes IGF binding proteins, which specifically bind IGF-I and IGF-II with high affinity ( a k, of 1.3~10” M.’ for IGF-I and 1.2~10’~ M’ for IGF-II respectively) and which may be capable of modulating glioma cell responsivenessto these peptides.

;yT 1 AEE~~~TSUMURA S,ZIKEDA %a . 1dep. Mcch.Engiiecr.Fat.Sci.Tech.KcioUniv., Yokohama,z Den.Biol.KcioUniv., Yokohama(&an) Purpos& Iniracellular free &lcium co&e&tion ([CaZ+]i)changesinduced by fluid flow were measuredin vascularendothelial cells (ECs) cultured from porcine aortae. Methods:ECs loaded Calcium Green-l/AM on a cover slip were mounted on a flow chamber. The working fluid was a HEPES-buffered saline supplementedwith 1 PM ATP.The relative fluorescent changes,which correspond to the [Ca2+]ichangelinearly, were measured with a confocal laser scanningmicroscope. Results:The fluid flow (flow rate: 0.26 ml/s&, shearstress:20 dyn/cm2) induced the transient, synchronized[CaZ+]iincreasein all cells and their +xk valuesof [caZ+]i were almost same. After the transient response,sustained[Ca2+]i increasewasobserved in several ECs (about 3040 % of cells), and it consistedof basal[Ca2+Ii level increaseand CaZ+spikes. Conclusions:‘l’be spatially averaged responseof [C&Ii showedthe gradual increase aspreviously reported. Theseresultsimply that fluid flow rate (or shearstress)is transduced into proportion of [CaZ+]ispiking cellsand/or spiking frequency in physiologicalcondition. 95