Bicarbonate-regulated Adenylyl Cyclase (sAC) Is a Sensor That Regulates pH-dependent V-ATPase Recycling

Abstract

Modulation of environmental pH is critical for the function of many biological systems. However, the molecular identity of the pH sensor and its interaction with downstream effector proteins remain poorly understood. Using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage, we now report a novel pathway for pH regulation linking the bicarbonate activated soluble adenylyl cyclase (sAC) to the vacuolar H+ATPase (V-ATPase). Clear cells of the epididymis and vas deferens contain abundant V-ATPase in their apical pole and are responsible for acidifying the lumen. Proton secretion is regulated via active recycling of V-ATPase. Here we demonstrate that this recycling is regulated by luminal pH and bicarbonate. sAC is highly expressed in clear cells, and apical membrane accumulation of V-ATPase is triggered by a sAC-dependent rise in cAMP in response to alkaline luminal pH. As sAC is expressed in other acid/base transporting epithelia, including kidney and choroid plexus, this cAMP-dependent signal transduction pathway may be a that cells to and pH. Modulation of environmental pH is critical for the function of many biological systems. However, the molecular identity of the pH sensor and its interaction with downstream effector proteins remain poorly understood. Using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage, we now report a novel pathway for pH regulation linking the bicarbonate activated soluble adenylyl cyclase (sAC) to the vacuolar H+ATPase (V-ATPase). Clear cells of the epididymis and vas deferens contain abundant V-ATPase in their apical pole and are responsible for acidifying the lumen. Proton secretion is regulated via active recycling of V-ATPase. Here we demonstrate that this recycling is regulated by luminal pH and bicarbonate. sAC is highly expressed in clear cells, and apical membrane accumulation of V-ATPase is triggered by a sAC-dependent rise in cAMP in response to alkaline luminal pH. As sAC is expressed in other acid/base transporting epithelia, including kidney and choroid plexus, this cAMP-dependent signal transduction pathway may be a that cells to and pH. soluble adenylyl cyclase (sAC) soluble adenylyl soluble adenylyl as a of cAMP a transduction pathway for cells to in as as the and pH sAC is adenylyl is to regulation by proteins is activated by bicarbonate and is in soluble and of sAC is to adenylyl in may be a for the bicarbonate regulation of cAMP in many biological is highly expressed in is to the cAMP that and for and are the are in a by pH of and a bicarbonate of that a of cells, the clear cells, are in the acidification of the Clear cells of the V-ATPase in their apical and are responsible for the of secretion in the vas Proton secretion by clear cells in a to kidney cells, clear cells their of secretion via V-ATPase recycling and the apical membrane cells, as as cells in the in V-ATPase and in apical with in secretion cells their of secretion in response to in the pH of their However, the molecular this response remain the we sAC a in the V-ATPase recycling that in and cells by and and in a as we to a in the of the by and are in identity of by of the for of the of the in a and the with pH epididymis and the and and as a the and of sAC and the a of and male with and via the with by a and as and male reproductive tract and by in the the of the V-ATPase and the and of of the male with as vas deferens the with a to a in the to the to the a of a of sperm with to pH as to the a of to in the of as the of the the luminal for for the of to the of vas deferens and and by in a and for in pH and in vas deferens and for of cells by for a of cells of epididymis by in and for V-ATPase of with as for V-ATPase and of of V-ATPase in the apical of clear cells the of of apical in the apical membrane including for and the by the of apical membrane and for that Using a and cells in are as in with of a as in the of pH and for and in the and cells by and in sAC and the of other clear the of this a the of sAC a the molecular of the in the sAC by a in clear cells, which by their for the V-ATPase in cells, as of sAC is expressed in the is in cells of of and and and with adenylyl cyclase in kidney Proton secretion is a function of clear cells and of of and sAC is highly expressed in cells of and epithelia, a in acid/base in for a of sAC in we the epididymis as a model in the of sAC in kidney of the and and of and of which by V-ATPase sAC and by in the with a of in to pH for and the luminal pH of clear cells, by their for a with cells and for and V-ATPase that a of contain that the is luminal pH of luminal pH V-ATPase recycling the pH of V-ATPase is the apical and and for that the with that a of V-ATPase is in the the alkaline luminal pH of V-ATPase is in apical and and in of V-ATPase and is a of V-ATPase clear cells to luminal pH with cells to pH of of V-ATPase with and the of V-ATPase that of V-ATPase to the apical membrane alkaline luminal pH. by of V-ATPase luminal pH and its in apical pH of the of a in the apical membrane of V-ATPase alkaline pH with pH apical V-ATPase via and in V-ATPase in the that clear cells to in luminal pH by a of V-ATPase the and apical apical membrane V-ATPase accumulation is a to luminal pH to its recycling luminal and epididymis with the luminal pH of of clear cells with Clear cells by their for V-ATPase the apical of clear cells, which is in in for and V-ATPase that a of in the contain V-ATPase and of luminal pH and the apical of clear cells epididymis with pH and for V-ATPase and V-ATPase is the apical membrane and of with and of the apical of clear cells epididymis with pH V-ATPase is in apical and in which are in the of of and the as a apical and the and V-ATPase in clear cells pH and pH luminal pH V-ATPase is in and are in apical luminal pH V-ATPase is in apical and are the the of luminal pH the of of apical clear cells for are expressed as and and the of that cAMP secretion in a of acidifying cells and the of many recycling membrane including and the of V-ATPase by luminal pH of with the cAMP for V-ATPase in the apical and of and that cAMP a of V-ATPase to the apical luminal pH of and sAC in V-ATPase the apical of clear cells to luminal and for V-ATPase and pH V-ATPase is in apical and with is the luminal pH. pH V-ATPase in the and of the apical are the alkaline luminal pH of with pH by are that cAMP the apical of V-ATPase. with pH the sAC V-ATPase is apical and of V-ATPase with that of sAC the alkaline of V-ATPase apical with pH by the of V-ATPase is in apical and with is that a of V-ATPase apical sAC with pH which apical of V-ATPase is that to the apical of V-ATPase by alkaline pH. to the the apical membrane and the of the for the V-ATPase sAC is activated by bicarbonate we the of in the and cAMP-dependent V-ATPase is the in biological and the and with and is by Clear cells of the and we that the the of secretion in vas deferens that bicarbonate via in response to alkaline luminal pH in of cAMP in response to sAC accumulation of V-ATPase in the apical in with pH in the of for to bicarbonate via clear cells of apical and V-ATPase in the and that bicarbonate via is in the of the V-ATPase the apical membrane alkaline luminal pH. of to the luminal for the of the in the of a of V-ATPase in apical V-ATPase the of the the apical V-ATPase of cAMP that cAMP of the in the V-ATPase sAC in this and cAMP-dependent we the of luminal alkaline pH V-ATPase of of the to soluble adenylyl cyclase and we sAC sAC by with of and with to pH in the of the sAC for a of V-ATPase apical and in a to that pH with of V-ATPase with that of sAC the alkaline response with of for the of the and in the of a of V-ATPase apical that clear cells their to to cAMP the of by which clear cells the V-ATPase to the apical membrane as that pH in the of of V-ATPase luminal pH that is of V-ATPase demonstrate that of V-ATPase apical by to of of by sAC in the of the of of with sAC as a function of in the of and in the of in the of in the of we clear cells the to to in luminal bicarbonate in to in pH. of in luminal bicarbonate pH V-ATPase and recycling with to pH a bicarbonate and with Clear cells to luminal bicarbonate apical apical membrane of the with clear cells to the pH in of bicarbonate and sAC the V-ATPase apical of the by the for the of the cells the apical membrane a in apical in the of bicarbonate and with the of bicarbonate and and that sAC the V-ATPase and of luminal bicarbonate V-ATPase the apical of clear cells with pH pH bicarbonate in the of for V-ATPase and V-ATPase is apical and of with in the of are and the V-ATPase is in apical a of the of and V-ATPase is apical and of sAC the apical V-ATPase and the of V-ATPase in by the by by the for the of the cells the apical pole for the cells to epididymis cells by is in the of bicarbonate in the of bicarbonate the pH the of the sAC the is with the bicarbonate that the V-ATPase the apical are expressed as that the of the V-ATPase in cells of the the clear cells, is to luminal pH. alkaline luminal the V-ATPase is in apical and is recycling and the apical that of bicarbonate is for the alkaline response and that cAMP apical of V-ATPase to that by alkaline pH. which are of the soluble adenylyl the V-ATPase sAC as the sensor responsible for the V-ATPase that of luminal by in pH in clear cells, to of bicarbonate sAC and cAMP which in to the accumulation of V-ATPase in apical responsible for the apical of V-ATPase remain and be to the downstream proteins in this Clear cells the to to in luminal bicarbonate to of bicarbonate the apical and in bicarbonate by sAC for the apical of bicarbonate be which is expressed in the apical membrane of clear cells that of cells to a of by bicarbonate the that bicarbonate secretion to this that the luminal of the epididymis is and a bicarbonate the of acidification in this that clear cells, which of the V-ATPase in their apical are in luminal acidification and that secretion by cells is of bicarbonate by the of V-ATPase in the in the of is in with of secretion that clear cells to a rise in luminal bicarbonate bicarbonate by their of the pH of the to its of sAC in clear cells with cells for a interaction and clear of sAC in kidney and cells the and and cells the the V-ATPase of sAC in as as the sAC in other acid/base transporting including the choroid that the sAC-dependent cAMP pathway may a cells to their of is the of the of sAC in in a and the pH and sAC is expressed in many biological in acid/base we that this cAMP pathway may a cells to and the pH of their molecular the response of cells to in the pH of their remain of a pathway that be to many biological systems. soluble adenylyl cyclase (sAC) soluble adenylyl soluble adenylyl as a of cAMP a transduction pathway for cells to in as as the and pH sAC is adenylyl is to regulation by proteins is activated by bicarbonate and is in soluble and of sAC is to adenylyl in may be a for the bicarbonate regulation of cAMP in many biological systems. soluble adenylyl cyclase soluble adenylyl cyclase sAC is highly expressed in is to the cAMP that and for and are the are in a by pH of and a bicarbonate of that a of cells, the clear cells, are in the acidification of the Clear cells of the V-ATPase in their apical and are responsible for the of secretion in the vas Proton secretion by clear cells in a to kidney cells, clear cells their of secretion via V-ATPase recycling and the apical membrane cells, as as cells in the in V-ATPase and in apical with in secretion cells their of secretion in response to in the pH of their However, the molecular this response remain the we sAC a in the V-ATPase recycling that in and cells by and and in a as we to a in the of the by and are in identity of by of the for of the of the in a and the with pH epididymis and the and and as a the and of sAC and the a of and male with and via the with by a and as and male reproductive tract and by in the the of the V-ATPase and the and of of the male with as vas deferens the with a to a in the to the to the a of a of sperm with to pH as to the a of to in the of as the of the the luminal for for the of to the of vas deferens and and by in a and for in pH and in vas deferens and for of cells by for a of cells of epididymis by in and for V-ATPase of with as for V-ATPase and of of V-ATPase in the apical of clear cells the of of apical in the apical membrane including for and the by the of apical membrane and for that Using a and cells in are as in with of a as in the of pH and for and cells by and and in a as we to a in the of the by and are in identity of by and the with pH epididymis and the and and as a the and of sAC and the a of and male with and via the with by a and as and male reproductive tract and by in the the of the V-ATPase and the and of of the male with as vas deferens the with a to a in the to the to the a of a of sperm with to pH as to the a of to in the of as the of the the luminal for for the of to the of vas deferens and and by in a and for in pH and in vas deferens and for of cells by for a of cells of epididymis by in and for V-ATPase of with as for V-ATPase and of of V-ATPase in the apical of clear cells the of of apical in the apical membrane including for and the by the of apical membrane and for that Using a and cells in are as in with of a as in the of pH and for and in the and cells by and in sAC and the of other clear the of this a the of sAC a the molecular of the in the sAC by a in clear cells, which by their for the V-ATPase in cells, as of sAC is expressed in the is in cells of of and and and with adenylyl cyclase in kidney Proton secretion is a function of clear cells and of of and sAC is highly expressed in cells of and epithelia, a in acid/base in for a of sAC in we the epididymis as a model by in the with a of in to pH for and the luminal pH of clear cells, by their for a with cells and for and V-ATPase that a of contain that the is 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by Clear cells of the and we that the the of secretion in vas deferens that bicarbonate via in response to alkaline luminal pH in of cAMP in response to sAC accumulation of V-ATPase in the apical in with pH in the of for to bicarbonate via clear cells of apical and V-ATPase in the and that bicarbonate via is in the of the V-ATPase the apical membrane alkaline luminal pH. of to the luminal for the of the in the of a of V-ATPase in apical V-ATPase the of the the apical V-ATPase of cAMP that cAMP of the sAC in the V-ATPase sAC in this and cAMP-dependent we the of luminal alkaline pH V-ATPase of of the to soluble adenylyl cyclase and we sAC sAC by with of and with to pH in the of the sAC for a of V-ATPase apical and in a to that pH with of V-ATPase with that of sAC the alkaline response with of for the of the and in the of a of V-ATPase apical that clear cells their to to cAMP the of by which clear cells the V-ATPase to the apical membrane as that pH in the of of V-ATPase luminal pH that is of V-ATPase demonstrate that of V-ATPase apical by to of the of we clear cells the to to in luminal bicarbonate in to in pH. of in luminal bicarbonate pH V-ATPase and recycling with to pH a bicarbonate and with Clear cells to luminal bicarbonate apical apical membrane of the with clear cells to the pH in of bicarbonate and sAC the V-ATPase apical of the by the for the of the cells the apical membrane a in apical in the of bicarbonate and with the of bicarbonate and and that sAC the V-ATPase and that the of the V-ATPase in cells of the the clear cells, is to luminal pH. alkaline luminal the V-ATPase is in apical and is recycling and the apical that of bicarbonate is for the alkaline response and that cAMP apical of V-ATPase to that by alkaline pH. which are of the soluble adenylyl the V-ATPase sAC as the sensor responsible for the V-ATPase that of luminal by in pH in clear cells, to of bicarbonate sAC and cAMP which in to the accumulation of V-ATPase in apical responsible for the apical of V-ATPase remain and be to the downstream proteins in this Clear cells the to to in luminal bicarbonate to of bicarbonate the apical and in bicarbonate by sAC for the apical of bicarbonate be which is expressed in the apical membrane of clear cells that of cells to a of by bicarbonate the that bicarbonate secretion to this that the luminal of the epididymis is and a bicarbonate the of acidification in this that clear cells, which of the V-ATPase in their apical are in luminal acidification and that secretion by cells is of bicarbonate by the of V-ATPase in the in the of is in with of secretion that clear cells to a rise in luminal bicarbonate bicarbonate by their of the pH of the to its of sAC in clear cells with cells for a interaction and clear of sAC in kidney and cells the and and cells the the V-ATPase of sAC in as as the sAC in other acid/base transporting including the choroid that the sAC-dependent cAMP pathway may a cells to their of is the of the of sAC in in a and the pH and sAC is expressed in many biological in acid/base we that this cAMP pathway may a cells to and the pH of their molecular the response of cells to in the pH of their remain of a pathway that be to many biological systems. that the of the V-ATPase in cells of the the clear cells, is to luminal pH. alkaline luminal the V-ATPase is in apical and is recycling and the apical that of bicarbonate is for the alkaline response and that cAMP apical of V-ATPase to that by alkaline pH. which are of the soluble adenylyl the V-ATPase sAC as the sensor responsible for the V-ATPase that of luminal by in pH in clear cells, to of bicarbonate sAC and cAMP which in to the accumulation of V-ATPase in apical responsible for the apical of V-ATPase remain and be to the downstream proteins in this Clear cells the to to in luminal bicarbonate to of bicarbonate the apical and in bicarbonate by sAC for the apical of bicarbonate be which is expressed in the apical membrane of clear cells that of cells to a of by bicarbonate the that bicarbonate secretion to this that the luminal of the epididymis is and a bicarbonate the of acidification in this that clear cells, which of the V-ATPase in their apical are in luminal acidification and that secretion by cells is of bicarbonate by the of V-ATPase in the in the of is in with of secretion that clear cells to a rise in luminal bicarbonate bicarbonate by their of the pH of the to its of sAC in clear cells with cells for a interaction and clear of sAC in kidney and cells the and and cells the the V-ATPase of sAC in as as the sAC in other acid/base transporting including the choroid that the sAC-dependent cAMP pathway may a cells to their of is the of the of sAC in in a and the pH and sAC is expressed in many biological in acid/base we that this cAMP pathway may a cells to and the pH of their molecular the response of cells to in the pH of their remain of a pathway that be to many biological systems. the of