apyrase and lucifer-yellow

Gap junctions and/or paracrine mediators, such as ATP, mediate intercellular communication (IC) in non-excitable cells. This study investigates the contribution of gap junctions toward IC during propagation of Ca(2+) waves in cultured bovine corneal endothelial cells (BCEC) elicited by applying a point mechanical stimulus to a single cell in a confluent monolayer. Changes in [Ca(2+)](i) were visualized using the fluorescent dye Fluo-4. The area reached by the Ca(2+) wave, called the active area (AA), was determined as a measure of efficacy of IC. RT-PCR and Western blotting showed expression of Cx43, a major form of connexin, in BCEC. In scrape-loading (using lucifer yellow) and fluorescence recovery after photobleaching (FRAP; using carboxyfluorescein) protocols, significant dye transfer of the hydrophilic dyes was evident indicating functional gap junctional IC (GJIC) in BCEC. Gap27 (300 microM), a connexin mimetic peptide that blocks gap junctions formed by Cx43, reduced the fluorescence recovery in FRAP experiments by 19%. Gap27 also reduced the active area of the Ca(2+) wave induced by point mechanical stimulation from 73,689 microm(2) to 26,936 microm(2), implying that GJIC contribution to the spread of the wave is at least approximately 63%. Inhibitors of ATP-mediated paracrine IC (PIC), such as a combination of apyrase VI and apyrase VII (5U/ml each; exogenous ATPases), suramin (200 microM; P2Y antagonist), or Gap26 (300 microM; blocker of Cx43 hemichannels) reduced the active area by 91%, 67%, and 55%, respectively. Therefore, estimating the contribution of GJIC from the residual active area after PIC inhibition appears to suggest that GJIC contributes no more than approximately 9% towards the active area of the Ca(2+) wave. Gap27 did not affect the enhancement in active area induced by ARL-67156 (200 microM, ectonucleotidase inhibitor), ATP release induced by point mechanical stimulation, and zero [Ca(2+)](o)-induced lucifer yellow uptake, indicating that the peptide has no influence on PIC. Exposure to Gap27 in the presence of PIC inhibitors led to a significant further inhibition of the Ca(2+) wave. The finding that the residual active area after inhibition of PIC by apyrases was much smaller than the reduction of the active area by Gap27, provides evidence for interaction between GJIC and PIC. These findings together suggest that functional gap junctions are present in BCEC, that both GJIC and PIC contribute significantly to IC, and that the

Topics: Adenosine Triphosphate; Animals; Apyrase; Calcium; Calcium Signaling; Cattle; Cell Communication; Cells, Cultured; Connexin 43; Connexins; Endothelial Cells; Endothelium, Corneal; Eye Proteins; Fluorescent Dyes; Gap Junctions; Immunohistochemistry; Isoquinolines; Oligopeptides; Paracrine Communication; Physical Stimulation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Suramin

Enteric glia are important participants in information processing in the enteric nervous system. However, intercellular signaling mechanisms in enteric glia remain largely unknown. We postulated that intercellular calcium waves exist in enteric glia. Primary cultures of enteric glia were isolated from neonatal guinea pig taenia coli. Intracellular calcium in individual cells was quantified with fura-2 AM microfluorimetry. Single-cell stimulation was performed with a micromanipulator-driven glass pipette. Data were expressed as mean +/- SEM and analyzed by Student's t-test. Mechanical stimulation of a single enteric glial cell resulted in an increase in intracellular calcium, followed by concentric propagation to 36% +/- 3% of neighboring cells. Intercellular calcium waves were blocked by depletion of intracellular calcium stores with thapsigargin (1 microM). Pretreatment of enteric glia with the phospholipase C inhibitor U73122 (1 microM) significantly decreased the percentage of cells responding to mechanical stimulation (6% +/- 4%), but had no effect on waves induced by microinjection of the inositol trisphosphate (67% +/- 13% vs. 60% +/- 4% for control). Antagonism of inositol trisphosphate receptor attenuated intercellular calcium waves induced by both mechanical stimulation and microinjection of inositol trisphosphate. Uncoupling of gap junctions with octanol or heptanol significantly inhibited intercellular calcium wave propagation. Pretreatment of enteric glia with apyrase partially attenuated intercellular calcium waves. Our data demonstrate that enteric glial cells are capable of transmitting increases in intracellular calcium to surrounding cells, and that intercellular calcium waves involve a sequence of intracellular and extracellular steps in which phospholipase C, inositol trisphosphate, and ATP play roles.

Topics: Adenosine Triphosphate; Alcohols; Animals; Animals, Newborn; Apyrase; Calcium; Calcium Signaling; Calcium-Binding Proteins; Cell Communication; Cells, Cultured; Digestive System; Digestive System Physiological Phenomena; DNA-Binding Proteins; Endothelin-3; Enteric Nervous System; Enzyme Inhibitors; Extracellular Space; Gap Junctions; Guinea Pigs; Inositol 1,4,5-Trisphosphate; Intracellular Fluid; Isoquinolines; Microfilament Proteins; Neuroglia; Signal Transduction; Type C Phospholipases

A change in intracellular Ca2+ is considered to be the common final signaling pathway through which renin secretion is governed. Therefore, information relating to the generation, control, and processing of Ca2+ signaling in juxtaglomerular cells (JG) will be critical for understanding JG cell behavior. In this study, we investigated the means by which JG cells harmonize their intracellular Ca2+ signals and explored the potential role of these mechanisms in renin secretion. Mechanical stimulation of a single JG cell initiated propagation of an intercellular Ca2+ wave to up to 11.9+/-4.1 surrounding cells, and this was prevented in the presence of the ATP-degrading enzyme, apyrase (1.7+/-0.7 cells), or by desensitization of purinergic receptors via pretreatment of cells with ATP (1.8+/-0.9 cells), thus implicating ATP as a mediator responsible for the propagation of intercellular Ca2+ signaling. Consistent with this, JG cells were demonstrated not to express the gap junction protein connexin43, and neither did they possess functional gap junction communication. Furthermore, massive mechanical stretching of JG cells elicited a 3-fold increase in ATP release. Administration of ATP into isolated perfused rat kidneys induced a rapid, potent, and persistent inhibition of renin secretion, together with a transient elevation of renal vascular resistance. ATP (1 mmol/L) caused up to 79% reduction of the renin secretion activated by lowering the renal perfusion flow (P<0.01). Taken together, our results indicate that under mechanical stimulation, ATP functions as a paracellular mediator to regulate renin secretion, possibly through modulating intra- and intercellular Ca2+ signals.

Topics: Adenosine Triphosphate; Animals; Apyrase; Calcium; Calcium Signaling; Cells, Cultured; Connexin 43; Glomerular Mesangium; Immunohistochemistry; In Vitro Techniques; Isoquinolines; Juxtaglomerular Apparatus; Kidney; Male; Rats; Rats, Wistar; Renin; Stress, Mechanical; Thapsigargin