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Role of intracellular Ca2+ in neuroendocrine control of growth hormone release in goldfish somatotropes
PI-34
P3-2
Role of intracellular Ca2’ in neuroendocrine control of growth hormone release in goldfish somatotropes.
Volume-activated eiYlux pathway for taurine (TAU) in reptilian renal brush-border membranes: is it a volumesensitive organic osmolyte-anion channel?
Yunker WK, and Chang JP Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9, CANADA. In goldfish, dopamine (DA) and gonadotropin-releasing hormone (GnRH) stimulate growth hormone (GH) release from pituitary somatotropes by CAMP/protein kinase A (PKA) and protein kinase C (PKC) signal transduction cascades respectively. These GH responses are dependent upon the availability of extracellular Ca2’. Somatostatin (SRIF14) is a regulatory peptide capable of reducing basal, as well as inhibiting GnRH- and DA-stimulated GH release. Using the Ca*‘-sensitive, fluorescent dye, Fura-2, the role of [Ca”]i during GnRH- and DA-stimulation, as well as during SRIF suppression of basal, and SRIF inhibition of ligand-stimulated GH release was examined both spatially and temporally in single identified somatotropes. A DA Dl agonist, SKF38393, and GnRH both increased [Ca”]i. Responses to both stimulators could be observed in the same cell, indicating the presence of both DA and GnRH receptors in at least some somatotropes. Preliminary results indicate that SRIF does not alter basal [Ca2’]i levels, but may prevent G&H-stimulated increases in [Ca*‘]i. These observations suggest an important role for [Ca*‘]i in regulated GH release. The relationships between [Ca2’]i fluctuations at the single cell level and GH release from populations of pituitary cells on comparable time scales are currently being investigated.
Benvaiati S Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73 190, USA. 3H-TAU efflux from purified brush-border membrane vesicles (BBMV) isolated from the kidney of garter snakes (7hamnophis spp.) was measured by a rapid filtration technique at 22°C. TAU efflux in isoosmotic medium was slow but could be significantly stimulated by osmotic swelling. TAU eflux was not dependent on Na’ or Cl-, showed a linear dependence on TAU concentrations (1 PM 30 mM), did not exhibit fruns stimulation, and preferred anionic TAU. The observed characteristics of the TAU efflux pathway in reptilian renal BBMV were similar to those of a swelling-activated organic osmolyte-anion channel found in many vertebrate cell types. The sensitivity of the reptilian volume-activated TAU efflux pathway to several inhibitors of At 250 PM, only the chloride channels was examined. stilbene DIDS significantly reduced hypoosmolality-activated TAU emux (67.2 + 7.5% of control). Other inhibitors (NPPB, 9AC, DPC, flufenamate, niflumic acid, SITS) were without effects. This suggests that one of the TAU et&x pathways in reptilian renal BBMV involves the band 3 anion exchanger functioning as a channel mediating TAU fluxes. P3-3
Characterization of a Na+-H+ exehanger from posterior gills of the estuarine crab Chamagnathm gramdata.
P3-1
Identification and isolation of peanut lectin binding cells in rainbow trout freshwater fish gill epithelium. SoDhia Adamia and Greg Goss, Dept. of Biol. Sciences, U. of Alberta, Edmonton, Alberta The freshwater fish gill epithelia (FFGE) is a heterogeneous system with respect to cell type and function. Mitochondria rich (MR) chloride cells are responsible for acid/base and ion transport regulation. Peanut lectin, which binds specifically to terminal D-galactosyl residues on glycosylated membrane proteins, has been used to identify P-type MR cells in the cortical collecting duct of mammalian kidneys. Using fluorescent probes (PNA-FITC 40 pg/ml, PNA-TRITC 40 pgiml) we identified a specific PNA-binding cell type in trypsin-dispersed gill cells of rainbow trout (Oncorhynchus mykiss). In gill epithelia, the mitochondrial stain DASPMI (40 pg/ml) can be used to identify MR cells. Using PNA-FITCYDASPMI doublestaining we identified the PNA-binding cell as a type of MR cell of the FFGE.
Bianchini A’, Rebouqas N2 and Malnic G2 ‘DCF, Funda@o Universidade do Rio Grande, Rio Grande, RS, ‘ICB, Universidade de Sb Paulo, Sb Paulo, SP, Brazil. C. gramdata is an eurihaline crab from salt marshes of Southern Brazil and Argentina. In the present work, the presence of Na+H’ exchanger in the posterior gills of diluted media acclimated crabs was analyzed. For this, a half platelet of posterior gill was fozd to a glass with poly-D-lysine and mounted in a bath chamber. Activity of the exchanger was determined after estimation of the intracellular pH @Hi) using a quantitative fluorescence system for radiometric studies (PMT-4000) and the pH-sensitive dye BCECF. Under physiological condition (355 mEq.l-’ Na+), the pHi was 7.60 and its complete restoration was observed after a pulse of NH+4 (1OOmM). When alI Na* was replaced by N-methyl-D-glucamine in the bath solution, a very slow pHi restoration was observed. But, in the presence of low Na+ concentration (5-50 mEq.F’), the pHi restoration was fastened. Amiloride ( 1u5 M), EIPA (10”’ I@, and DMA (lo4 M) significantly inhibited the pHi restoration. These results indicate that posterior gills effectively possess an amiloride sensitive Na+-p exchanger, which is probably involved in transepithelial NaCl transport in C. grandafa. Further, as gill Nd,K+- ATPase, Nat-H+ exchanger showed a high affinity by Na+. Financial support: CNPq, FAPERGS, FAPESP
P3-2
Role of intracellular Ca2’ in neuroendocrine control of growth hormone release in goldfish somatotropes.
Volume-activated eiYlux pathway for taurine (TAU) in reptilian renal brush-border membranes: is it a volumesensitive organic osmolyte-anion channel?
Yunker WK, and Chang JP Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9, CANADA. In goldfish, dopamine (DA) and gonadotropin-releasing hormone (GnRH) stimulate growth hormone (GH) release from pituitary somatotropes by CAMP/protein kinase A (PKA) and protein kinase C (PKC) signal transduction cascades respectively. These GH responses are dependent upon the availability of extracellular Ca2’. Somatostatin (SRIF14) is a regulatory peptide capable of reducing basal, as well as inhibiting GnRH- and DA-stimulated GH release. Using the Ca*‘-sensitive, fluorescent dye, Fura-2, the role of [Ca”]i during GnRH- and DA-stimulation, as well as during SRIF suppression of basal, and SRIF inhibition of ligand-stimulated GH release was examined both spatially and temporally in single identified somatotropes. A DA Dl agonist, SKF38393, and GnRH both increased [Ca”]i. Responses to both stimulators could be observed in the same cell, indicating the presence of both DA and GnRH receptors in at least some somatotropes. Preliminary results indicate that SRIF does not alter basal [Ca2’]i levels, but may prevent G&H-stimulated increases in [Ca*‘]i. These observations suggest an important role for [Ca*‘]i in regulated GH release. The relationships between [Ca2’]i fluctuations at the single cell level and GH release from populations of pituitary cells on comparable time scales are currently being investigated.
Benvaiati S Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73 190, USA. 3H-TAU efflux from purified brush-border membrane vesicles (BBMV) isolated from the kidney of garter snakes (7hamnophis spp.) was measured by a rapid filtration technique at 22°C. TAU efflux in isoosmotic medium was slow but could be significantly stimulated by osmotic swelling. TAU eflux was not dependent on Na’ or Cl-, showed a linear dependence on TAU concentrations (1 PM 30 mM), did not exhibit fruns stimulation, and preferred anionic TAU. The observed characteristics of the TAU efflux pathway in reptilian renal BBMV were similar to those of a swelling-activated organic osmolyte-anion channel found in many vertebrate cell types. The sensitivity of the reptilian volume-activated TAU efflux pathway to several inhibitors of At 250 PM, only the chloride channels was examined. stilbene DIDS significantly reduced hypoosmolality-activated TAU emux (67.2 + 7.5% of control). Other inhibitors (NPPB, 9AC, DPC, flufenamate, niflumic acid, SITS) were without effects. This suggests that one of the TAU et&x pathways in reptilian renal BBMV involves the band 3 anion exchanger functioning as a channel mediating TAU fluxes. P3-3
Characterization of a Na+-H+ exehanger from posterior gills of the estuarine crab Chamagnathm gramdata.
P3-1
Identification and isolation of peanut lectin binding cells in rainbow trout freshwater fish gill epithelium. SoDhia Adamia and Greg Goss, Dept. of Biol. Sciences, U. of Alberta, Edmonton, Alberta The freshwater fish gill epithelia (FFGE) is a heterogeneous system with respect to cell type and function. Mitochondria rich (MR) chloride cells are responsible for acid/base and ion transport regulation. Peanut lectin, which binds specifically to terminal D-galactosyl residues on glycosylated membrane proteins, has been used to identify P-type MR cells in the cortical collecting duct of mammalian kidneys. Using fluorescent probes (PNA-FITC 40 pg/ml, PNA-TRITC 40 pgiml) we identified a specific PNA-binding cell type in trypsin-dispersed gill cells of rainbow trout (Oncorhynchus mykiss). In gill epithelia, the mitochondrial stain DASPMI (40 pg/ml) can be used to identify MR cells. Using PNA-FITCYDASPMI doublestaining we identified the PNA-binding cell as a type of MR cell of the FFGE.
Bianchini A’, Rebouqas N2 and Malnic G2 ‘DCF, Funda@o Universidade do Rio Grande, Rio Grande, RS, ‘ICB, Universidade de Sb Paulo, Sb Paulo, SP, Brazil. C. gramdata is an eurihaline crab from salt marshes of Southern Brazil and Argentina. In the present work, the presence of Na+H’ exchanger in the posterior gills of diluted media acclimated crabs was analyzed. For this, a half platelet of posterior gill was fozd to a glass with poly-D-lysine and mounted in a bath chamber. Activity of the exchanger was determined after estimation of the intracellular pH @Hi) using a quantitative fluorescence system for radiometric studies (PMT-4000) and the pH-sensitive dye BCECF. Under physiological condition (355 mEq.l-’ Na+), the pHi was 7.60 and its complete restoration was observed after a pulse of NH+4 (1OOmM). When alI Na* was replaced by N-methyl-D-glucamine in the bath solution, a very slow pHi restoration was observed. But, in the presence of low Na+ concentration (5-50 mEq.F’), the pHi restoration was fastened. Amiloride ( 1u5 M), EIPA (10”’ I@, and DMA (lo4 M) significantly inhibited the pHi restoration. These results indicate that posterior gills effectively possess an amiloride sensitive Na+-p exchanger, which is probably involved in transepithelial NaCl transport in C. grandafa. Further, as gill Nd,K+- ATPase, Nat-H+ exchanger showed a high affinity by Na+. Financial support: CNPq, FAPERGS, FAPESP