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A J Cholewinski

Publications and source records attributed to A J Cholewinski.

9 recordsLinked to original sources
PubMed1995

Characterization of the 5-hydroxytryptamine2A receptor-activated cascade in rat C6 glioma cells.

We have investigated the identity and intracellular cascade of responses resulting from activation of the endogenous 5-hydroxytryptamine receptor in the C6 rat glioma cell line. Sequence analysis of reverse transcription-polymerase chain reaction products derived from C6 glioma cell messenger RNA revealed complete homology with a portion of the rat 5-hydroxytryptamine2A receptor. The binding of [3H]ketanserin to cell membranes demonstrated a significant correlation with the 5-hydroxytryptamine2A receptor in rat frontal cortex. On intact cells, 5-hydroxytryptamine stimulated a concentration-dependent increase in phosphatidyl inositide turnover and intracellular [Ca2+] mediated by 5-hydroxytryptamine2A receptors. In whole-cell patch-clamp recordings, 5-hydroxytryptamine induced an outward current mediated predominantly by K+ ions (reversal potential = -80 mV). Using caged molecules containing Ca2+ or inositol 1,4,5-trisphosphate in the patch electrode solution, we found that rapid photolytic release of Ca2+ and particularly inositol 1,4,5-trisphosphate within the cytosol induced an outward current with characteristics similar to those seen after application of 5-hydroxytryptamine. Comparison between differentiated and undifferentiated cells revealed significantly higher receptor density and maximal phosphoinositide response to 5-hydroxytryptamine in undifferentiated cells but the associated rise in [Ca2+]i and activation of an outward current was observed more frequently in differentiated cells. Prolonged exposure of the cells to 5-hydroxytryptamine led to a decrease in all responses and to the down-regulation of receptor number. We conclude that the rat C6 glioma cell expresses a 5-hydroxytryptamine2A receptor identical to that found in rat brain and that stimulation of the receptor in C6 cells leads to the activation of Ca2+ activated K+ channels via phosphoinositide hydrolysis and subsequent rise in cytosolic Ca2+ ion concentration. However, the contrasting effects of differentiation on receptor number and phosphoinositide response to 5-hydroxytryptamine compared to Ca2+ release and conductance change indicate that a complex relationship exists between the component parts of the receptor-activated cascade.

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PubMed1993

Calcium-mobilizing and electrophysiological effects of bradykinin on cortical astrocyte subtypes in culture.

The possible consequences of activating bradykinin (Bk) receptors on identified astrocyte subtypes derived from rat cortex have been investigated in terms of: 1) mobilization of intracellular Ca2+; and 2) electrophysiological response. Bk induced a rapid, transient rise in intracellular Ca2+ in 63% of cortical type-1-like astrocytes and 44% of type-2 astrocytes tested. Experiments involving Ca(2+)-free conditions suggested that the release occurred largely from internal stores in both astrocyte subtypes. Bk receptor activation resulted in an inward current in approximately 10% of each astrocyte subtype tested using the whole-cell patch clamp technique. The use of perforated patch recording confirmed a similar population of cells responsive to Bk. The Bk-induced current was associated with a decrease in membrane conductance. The reversal potential was close to the K+ equilibrium potential, consistent with the closure of K+ channels. The current demonstrated similar EC50 values and Hill coefficients and also a marked degree of desensitization for both astrocyte subtypes. Recordings from type-1-like astrocytes indicated that the receptor mediating the electrophysiological response was of the B2 subtype. The data were consistent with the functional translation of Bk receptor occupation to physiological responses in distinct sub-populations of cortical type-1-like and type-2 astrocytes.

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PubMed1991

Identification of B2 bradykinin binding sites on cultured cortical astrocytes.

Bradykinin was found to bind to specific high-affinity sites in cultured cortical astrocytes from rat brain, and this binding appeared to be specific for the B2 bradykinin receptor subtype. Nonlinear regression analysis of saturation experiments using a computer programme revealed a single KD of 16.6 +/- 2.6 nM and a Bmax of 352.2 +/- 30.7 fmol/mg of protein. These results indicate that astrocytes possess bradykinin receptors and that these are predominantly of the B2 subtype.

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PubMed1988

A phosphoinositide-linked peptide response in astrocytes: evidence for regional heterogeneity.

A phosphoinositide-linked peptide response in cultured rat astrocytes was studied by measuring the accumulation of [3H]inositol phosphates in the presence of lithium. Cultures derived from cortex, cerebellum and spinal cord each showed a unique pattern or degree of stimulation to a panel of neuropeptides. Cortical and cerebellar astrocytes were similar, responding to bradykinin, oxytocin, vasopressin, eledoisin and neurokinin beta, whereas spinal cord astrocytes were stimulated by substance P, bradykinin, eledoisin, and neurokinins alpha and beta. These observations are evidence in favour of regional specialisations of astrocytes which may respond uniquely to peptides released by particular populations of neurons.

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PubMed1988

Astrocytes from forebrain, cerebellum, and spinal cord differ in their responses to vasoactive intestinal peptide.

Astrocytes from cortex, cerebellum, and spinal cord responded to isoproterenol and vasoactive intestinal peptide (VIP) with increases in intracellular cyclic AMP levels. The response to VIP was as great as that to isoproterenol in cortical astrocytes (180-fold and 185-fold, respectively), and the effect of VIP in combination with isoproterenol was partially additive. Spinal cord astrocytes also responded to VIP and isoproterenol with equal potency (seven- to ninefold and eight- to 13-fold, respectively), but the level of response was much smaller than in cortex. Spinal cord astrocytes were synergistic in their response to VIP and isoproterenol. The response to VIP was lowest in cerebellar astrocytes (only threefold), and no additivity was observed when VIP was added together with isoproterenol. A small response to alpha-melanocyte stimulating hormone (alpha-MSH) was also observed in cortex and cerebellum, but not in spinal cord. Somatostatin inhibited the response to isoproterenol in cortex and cerebellum, but had no effect in spinal cord. The results from the above study show that astrocytes obtained from these three regions of the rat CNS express quite different responses to VIP and alpha-MSH and further point to possible astrocyte heterogeneity.

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PubMedDate not supplied

5-Hydroxytryptamine-evoked [Ca2+]i oscillations in rat C6 glioma cells.

We have investigated the modulation of the intracellular calcium concentration ([Ca2+]i) in rat C6 glioma cells following their activation by the agonists 5-hydroxytryptamine.HCl (5-HT) and bradykinin, using single cell imaging of [Ca2+]i with the calcium-sensitive dye Fura-2. The majority of the signals observed involved release of calcium from intracellular stores, and after prolonged application of 5-HT, but not bradykinin, the cells exhibited oscillations in [Ca2+]i levels. These calcium oscillations were dependent on the presence of extracellular calcium, and were unaffected by the calcium channel antagonists nifedipine and verapamil. Caffeine, which in other cell types is able to release calcium from inositol trisphosphate-insensitive stores, had very little effect on [Ca2+]i levels in C6 cells. On the other hand, bradykinin, although able to elevate [Ca2+]i probably by acting via the B2-receptor subtype, was unable to induce any calcium oscillations in these cells.

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