- Email: [email protected]
Angiotensin II receptor internalisation and cycling in the rat adrenal zona glomerulosa cell
146
ANGIOTENSIN II RECEPTOR INTERNALISATION AND CYCLING 1N THE RA F ADRENAL ZONA GLOMERULOSA CELL G P Vlnson, M M Ho, R Teja & J R Puddeloot, Department ol Biochemistry, Queen Mary & Westfleld College, London E1 4NS Indirect evidence exists Ior cellular translx)rt and mtemahsatlon ol the ang]otensm ll AT 1 receptor Using a recently developed monockmal ant:btxty, 6313/G2, to a unique sequence in the extracellular N-terminal domain of the AT l receptor we have Iocahsed the receptor :n target cells, and have thus obtmned direct evidence for receptor cycling and mtemahsatmn Rat adrenal zona giomerulosa cells, obtmned by collagenase dmpersal, were layered on to microscope shdes, fixed with 3 7% (v/v) formaldehyde, and AT l receptors were locahsed by fluorescent lmmun(~ cytochem:stry In unstlmulated cells, the signal is predominantly c)toplasmlc However, when prev:able cells were first m<.ubated with 6313/G2 :n mtxilfied RPMI-1640 medmm prior to immunocytochemlstry, the receptor was mostly locahsed m clumped patches m the plasma membrane Incubation of glomerulosa cells with 6313/G2 also stimulates aldosterone pr(xtuct:on approximately 2fold, similar to angmtensm II amlde at 100 nmol/I, though the actmns of 6313/G2 and All are not add:ave, although those of 6313/G2 and ACTH(1-24), lOpmtd/i, or K + runs at 5 9mmol/l, may be Studies using 1251-labelled AII show that 6313/G2 has no effect on AII binding to receptor The data suggest that the receptor is continuously cycled between the plasma membrane and mtracellular locahsat:ons Stimulation of steroidogenes:s can be achieved by anchonng the receptor m the membrane by binding to antltxxiy 6313/G2
DETECTION OF RENIN AND ANGIOTENSIN-CONVERTING ENZYME IN MITOCHONDRIA OF THE RAT ADRENAL CORTEX J Peters, S Resch, S Bachmann, and E Hackenthal Dept of Pharmacology and Dept of Anatomy, Umvermty of He,delberg, Germany Obmctw~ The presence of ren,n has been demonstrated in the rat adrenal gland To ,nvest,gate the question of possible mtracellular functmns of renm ,n the adrenal gland we analyzed the subcellular d,stnbutmn of adrenal renm by means of ,mmunocytochemmtryelectronmmroscopy us,ng an ant,-renm antibody and analyzed angmtensm II (ANGII) binding to the different ,ntracellular compartements Results Surprisingly, remn-epec,fic ,mmunogold staining was found not only w,thln the known cytosohc ves,cles, but also w,thm distinct mtramltochondr:al dense bodies ,n a number of cells of the inner zone glomerulosa Significant stam,ng at this site was also found w,th an antibody specdm for ang,otensm converting enzyme (ACE), but not w,th antibodies against the lysosomal marker enzymes, such as cathepsm B and D, or w,th a nonspec,f,c ant,serum In add,t,on, mdmated ANG II was found to bind to membranes derived from punf:ed m,tochondnal fractmns Th,s b,ndmg was mh,bited m a dose dependent manner by nonlabeled ANG II (0 l n M to 20nM}, as well as by the ANGreceptor antagonist DUP 753 No binding was observed with membranes of the nuclear fract,ons Conclusmn The presence of renm, ACE and ANG-bmdmg sites w,thm adrenocort,cal m,tochondna suggests the exmtence of an mtracellular renm-angmtensm system ,n the adrenal gland Thin system may be revolved ,n m,tochondnal stero,dogenesm
ANGIOTENSIN II RECEPTOR INTERNALISATION AND CYCLING 1N THE RA F ADRENAL ZONA GLOMERULOSA CELL G P Vlnson, M M Ho, R Teja & J R Puddeloot, Department ol Biochemistry, Queen Mary & Westfleld College, London E1 4NS Indirect evidence exists Ior cellular translx)rt and mtemahsatlon ol the ang]otensm ll AT 1 receptor Using a recently developed monockmal ant:btxty, 6313/G2, to a unique sequence in the extracellular N-terminal domain of the AT l receptor we have Iocahsed the receptor :n target cells, and have thus obtmned direct evidence for receptor cycling and mtemahsatmn Rat adrenal zona giomerulosa cells, obtmned by collagenase dmpersal, were layered on to microscope shdes, fixed with 3 7% (v/v) formaldehyde, and AT l receptors were locahsed by fluorescent lmmun(~ cytochem:stry In unstlmulated cells, the signal is predominantly c)toplasmlc However, when prev:able cells were first m<.ubated with 6313/G2 :n mtxilfied RPMI-1640 medmm prior to immunocytochemlstry, the receptor was mostly locahsed m clumped patches m the plasma membrane Incubation of glomerulosa cells with 6313/G2 also stimulates aldosterone pr(xtuct:on approximately 2fold, similar to angmtensm II amlde at 100 nmol/I, though the actmns of 6313/G2 and All are not add:ave, although those of 6313/G2 and ACTH(1-24), lOpmtd/i, or K + runs at 5 9mmol/l, may be Studies using 1251-labelled AII show that 6313/G2 has no effect on AII binding to receptor The data suggest that the receptor is continuously cycled between the plasma membrane and mtracellular locahsat:ons Stimulation of steroidogenes:s can be achieved by anchonng the receptor m the membrane by binding to antltxxiy 6313/G2
DETECTION OF RENIN AND ANGIOTENSIN-CONVERTING ENZYME IN MITOCHONDRIA OF THE RAT ADRENAL CORTEX J Peters, S Resch, S Bachmann, and E Hackenthal Dept of Pharmacology and Dept of Anatomy, Umvermty of He,delberg, Germany Obmctw~ The presence of ren,n has been demonstrated in the rat adrenal gland To ,nvest,gate the question of possible mtracellular functmns of renm ,n the adrenal gland we analyzed the subcellular d,stnbutmn of adrenal renm by means of ,mmunocytochemmtryelectronmmroscopy us,ng an ant,-renm antibody and analyzed angmtensm II (ANGII) binding to the different ,ntracellular compartements Results Surprisingly, remn-epec,fic ,mmunogold staining was found not only w,thln the known cytosohc ves,cles, but also w,thm distinct mtramltochondr:al dense bodies ,n a number of cells of the inner zone glomerulosa Significant stam,ng at this site was also found w,th an antibody specdm for ang,otensm converting enzyme (ACE), but not w,th antibodies against the lysosomal marker enzymes, such as cathepsm B and D, or w,th a nonspec,f,c ant,serum In add,t,on, mdmated ANG II was found to bind to membranes derived from punf:ed m,tochondnal fractmns Th,s b,ndmg was mh,bited m a dose dependent manner by nonlabeled ANG II (0 l n M to 20nM}, as well as by the ANGreceptor antagonist DUP 753 No binding was observed with membranes of the nuclear fract,ons Conclusmn The presence of renm, ACE and ANG-bmdmg sites w,thm adrenocort,cal m,tochondna suggests the exmtence of an mtracellular renm-angmtensm system ,n the adrenal gland Thin system may be revolved ,n m,tochondnal stero,dogenesm