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Regulation of ion transport by chloride cells of euryhaline teleost gill epithelium: hormones, tonicity, and cytoskeleton
Abstracts / Comparative Biochemisto' and Physiology, Part A 126 (2000) SI-SI63
SIGNAL
TRANSDUCTION
PATHWAYS
INVOLVED
IN
REGULATION
S 101
OF KIDNEY
COLLECTING DUCT AQUAPORINS M a r p l e s D. School of Biomedical Sciences, University of Leeds, Leeds LS2 9NQ, England The renal collecting duct is the site of vasopressin-regulated water reabsorption, and determines how concentrated the urine produced by the kidney will be. Vasopressin increases the water permeability of the apical plasma membrane of the collecting duct principal cells by causing the exocytic insertion of specific water channels (aquaporin-2, AQP2) from a store in intracellular vesicles. This process is mediated by V2 receptors in the basolateral membrane of the cells, which activate adenylate cyclase. The production of cAMP activates protein kinase A, which phosphoryates a range of target proteins, including AQP2 itself. This is a key step in the regulated translocation of the channels to the plasma membrane. This process reaches a maximum over about half and hour. When vasopressin levels fall, AQP2 is removed from the membrane endocytically. The channels may then be available for recycling, although this remains a matter of debate, since there is also some evidence for degradation and/or excretion of the channels. In addition to the acute shuttling of AQP2 to and from the apical plasma membrane, there is a long-term modulation of collecting duct water permeability, which is brought about by changes in the total amount of AQP2 present in the cells. It is clear that vasopressin, acting via cAMP and a cAMP-responsive element in the promoter region of the AQP2 gene, is also a key factor in this long-term regulation. However additional, and probably more powerful, stimuli are also present, since (a) the effect of vasopressin can be overcome by water loading, and (b) chronic dehydration can induce increased AQP2 expression even when no vasopressin-induced shuttling is seen. The stimuli and signaling cascades involved in this process are currently being sought. Interstitial tonicity and urine flow rate do not appear to be significant factors, but there is indirect evidence that prostaglandins produced in response to dehydration do induce AQP2 expression. Water entering the cells through AQP2 in the apical plasma membrane leaves across the basolateral membrane through the related water channels AQP3 and AQP4. These channels are constitutively present in the plasma membrane, rather than being shuttled in response to vasopressin, but the amount of AQP3 appears to undergo a long-term modulation in response to vasopressin similar to that seen for AQP2. However, the changes in expression of these water channels are not always correlated, suggesting that a more complex pattern of signals may be involved in determining the expression of the basolateral water channels, as well as of AQP2. REGULATION OF ION TRANSPORT BY CHLORIDE CELLS OF EURYHALINE TELEOST GILL EPITHELIUM: H O R M O N E S , T O N I C I T Y , A N D C Y T O S K E L E T O N M a r s h a l l W . S . , D a b o r n K., D o y l e T., M c C o r m i c k S . D . 1 a n d S i n g e r T . D . 2 B i o l o g y D e p t . , St. F r a n c i s X a v i e r U n i v . , A n t i g o n i s h N S , C a n a d a , ~Conte A n a d r o m o u s F i s h R e s e a r c h L a b o r a t o r y , T u r n e r s F a l l s M A , U S A , 2Biol. D e p t . , U. W a t e r l o o , W a t e r l o o , O N , C a n a d a Euryhaline teleost fish readily adapt to fresh water (FW) or to seawater (SW) and in the process reverse ion transport direction across the gill and opercular epithelium, turning on Cl- secretion when acclimating to SW and shutting down this secretion during acclimation to FW. We identified by patch clamp a 7 pS anion channel in short term primary cultures of Fundulus heteroclitus chloride (C1-) cells that was similar to the mammalian Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The channel is inhibited by apically added DPC and NPPB, activated by cAMP in whole cells and by protein kinase A (PKA)+ATP in isolated membrane patches. A 5.7-kilobase cDNA isolated from killifish gill encodes a protein product (kfCFTR) that is 59% identical to human CFTR (T. Singer et al., Am. J. Physiol. 274:C715-C723, '98) and when expressed in Xenopus oocytes imparts an cAMP stimulatable C1- current. Hence there is in the apical membrane of chloride cells a CFTR C1 channel activated by PKA. CI- secretion is inhibited by K+-free saline and bumetanide, indicating basolateral location for a Na,K,C1 cotransporter (NaKCC). The time course of adaptation of killifish to SW involves a peak in plasma cortisol at 1h followed by a rise in plasma Na + at 3h, elevated plasma osmolality and enhanced expression of kfCFTR at 8h+ followed by increased CI- secretory rate (by opercular epithelia) at 24h+ such that plasma osmolality and ion content reach a new steady state by 7d. Mimicking hsperionic (above SW) conditions by added NaC1 on the basolateral side of opercular epithelia in vitro has little effect on CI secretion rate, but causes CI- cells to swell. The effect is blocked by basolateral bumetanide and exacerbated by apical DPC. We interpret the effect as an accumulation of ions via basolateral NaKCC, along with water, but with apical CFTR being rate limiting. Clonidine acts via c~2-adrenoceptors and enhance inositoltrisphosphate and Ca 2+ to rapidly inhibit C1secretion and acts at NaKCC, as clonidine blocks the hyperionic swelling response. Isoproterenol acts via ~-adrenergic receptors, cAMP and PKA to phosphorylate and activate apical CFTR, also preventing the hyperionic swelling response but by removing the rate limiting step. Hypertonic shock rapidly shrinks CI- cells and stimulates C1- secretion (Zadunaisky et al. J. Membr. Biol. 143:207-217, '95), while basolateral hypotonic shock swells C1- cells and inhibits CI- secretion apparently via inhibition of protein tyrosine kinase, as the effect is mimicked by genistein but not by its analog daidzein. Thus C1 cells respond strongly to rapid adrenergic and osmotic stimuli and during SW adaptation (mediated by cortisol and increased blood osmolality) augment kfCFTR expression to upregulate the rate limiting step of CI- secretion.
SIGNAL
TRANSDUCTION
PATHWAYS
INVOLVED
IN
REGULATION
S 101
OF KIDNEY
COLLECTING DUCT AQUAPORINS M a r p l e s D. School of Biomedical Sciences, University of Leeds, Leeds LS2 9NQ, England The renal collecting duct is the site of vasopressin-regulated water reabsorption, and determines how concentrated the urine produced by the kidney will be. Vasopressin increases the water permeability of the apical plasma membrane of the collecting duct principal cells by causing the exocytic insertion of specific water channels (aquaporin-2, AQP2) from a store in intracellular vesicles. This process is mediated by V2 receptors in the basolateral membrane of the cells, which activate adenylate cyclase. The production of cAMP activates protein kinase A, which phosphoryates a range of target proteins, including AQP2 itself. This is a key step in the regulated translocation of the channels to the plasma membrane. This process reaches a maximum over about half and hour. When vasopressin levels fall, AQP2 is removed from the membrane endocytically. The channels may then be available for recycling, although this remains a matter of debate, since there is also some evidence for degradation and/or excretion of the channels. In addition to the acute shuttling of AQP2 to and from the apical plasma membrane, there is a long-term modulation of collecting duct water permeability, which is brought about by changes in the total amount of AQP2 present in the cells. It is clear that vasopressin, acting via cAMP and a cAMP-responsive element in the promoter region of the AQP2 gene, is also a key factor in this long-term regulation. However additional, and probably more powerful, stimuli are also present, since (a) the effect of vasopressin can be overcome by water loading, and (b) chronic dehydration can induce increased AQP2 expression even when no vasopressin-induced shuttling is seen. The stimuli and signaling cascades involved in this process are currently being sought. Interstitial tonicity and urine flow rate do not appear to be significant factors, but there is indirect evidence that prostaglandins produced in response to dehydration do induce AQP2 expression. Water entering the cells through AQP2 in the apical plasma membrane leaves across the basolateral membrane through the related water channels AQP3 and AQP4. These channels are constitutively present in the plasma membrane, rather than being shuttled in response to vasopressin, but the amount of AQP3 appears to undergo a long-term modulation in response to vasopressin similar to that seen for AQP2. However, the changes in expression of these water channels are not always correlated, suggesting that a more complex pattern of signals may be involved in determining the expression of the basolateral water channels, as well as of AQP2. REGULATION OF ION TRANSPORT BY CHLORIDE CELLS OF EURYHALINE TELEOST GILL EPITHELIUM: H O R M O N E S , T O N I C I T Y , A N D C Y T O S K E L E T O N M a r s h a l l W . S . , D a b o r n K., D o y l e T., M c C o r m i c k S . D . 1 a n d S i n g e r T . D . 2 B i o l o g y D e p t . , St. F r a n c i s X a v i e r U n i v . , A n t i g o n i s h N S , C a n a d a , ~Conte A n a d r o m o u s F i s h R e s e a r c h L a b o r a t o r y , T u r n e r s F a l l s M A , U S A , 2Biol. D e p t . , U. W a t e r l o o , W a t e r l o o , O N , C a n a d a Euryhaline teleost fish readily adapt to fresh water (FW) or to seawater (SW) and in the process reverse ion transport direction across the gill and opercular epithelium, turning on Cl- secretion when acclimating to SW and shutting down this secretion during acclimation to FW. We identified by patch clamp a 7 pS anion channel in short term primary cultures of Fundulus heteroclitus chloride (C1-) cells that was similar to the mammalian Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The channel is inhibited by apically added DPC and NPPB, activated by cAMP in whole cells and by protein kinase A (PKA)+ATP in isolated membrane patches. A 5.7-kilobase cDNA isolated from killifish gill encodes a protein product (kfCFTR) that is 59% identical to human CFTR (T. Singer et al., Am. J. Physiol. 274:C715-C723, '98) and when expressed in Xenopus oocytes imparts an cAMP stimulatable C1- current. Hence there is in the apical membrane of chloride cells a CFTR C1 channel activated by PKA. CI- secretion is inhibited by K+-free saline and bumetanide, indicating basolateral location for a Na,K,C1 cotransporter (NaKCC). The time course of adaptation of killifish to SW involves a peak in plasma cortisol at 1h followed by a rise in plasma Na + at 3h, elevated plasma osmolality and enhanced expression of kfCFTR at 8h+ followed by increased CI- secretory rate (by opercular epithelia) at 24h+ such that plasma osmolality and ion content reach a new steady state by 7d. Mimicking hsperionic (above SW) conditions by added NaC1 on the basolateral side of opercular epithelia in vitro has little effect on CI secretion rate, but causes CI- cells to swell. The effect is blocked by basolateral bumetanide and exacerbated by apical DPC. We interpret the effect as an accumulation of ions via basolateral NaKCC, along with water, but with apical CFTR being rate limiting. Clonidine acts via c~2-adrenoceptors and enhance inositoltrisphosphate and Ca 2+ to rapidly inhibit C1secretion and acts at NaKCC, as clonidine blocks the hyperionic swelling response. Isoproterenol acts via ~-adrenergic receptors, cAMP and PKA to phosphorylate and activate apical CFTR, also preventing the hyperionic swelling response but by removing the rate limiting step. Hypertonic shock rapidly shrinks CI- cells and stimulates C1- secretion (Zadunaisky et al. J. Membr. Biol. 143:207-217, '95), while basolateral hypotonic shock swells C1- cells and inhibits CI- secretion apparently via inhibition of protein tyrosine kinase, as the effect is mimicked by genistein but not by its analog daidzein. Thus C1 cells respond strongly to rapid adrenergic and osmotic stimuli and during SW adaptation (mediated by cortisol and increased blood osmolality) augment kfCFTR expression to upregulate the rate limiting step of CI- secretion.