Contr. Nephrol., vol. 14, pp. 74-86 (Karger, Basel 1978)
Renal Adenylate Cyclase Systems H Osswald! and T.E. Northrup Abteilung Pharmakologie der Medizinischen Fakultat der RWfH Aachen, Aachen, and Department of Physiology and Biophysics and Division of Nephrology, Mayo Clinic and Mayo Foundation, Rochester, Minn.
Introduction Transepithelial movement of electrolytes and water is accomplished by controlling both the energy-consuming active transport steps and the permeability characteristics of the membranes across which transport is occurring. Energy for renal transport is basically derived from the hydrolysis of adenosine triphosphate (ATP). In the kidney this reaction is catalyzed by a variety of ATPases, predominantly Na-K-ATPase. There is no evidence that cyclic nucleotides are involved in regulating the activity of Na-K-ATPase in the kidney. Rather, cyclic nucleotides seem to be directly involved in regulating the transport characteristics of membranes in the nephron. These characteristics may involve permeability components or structures associated with various carriers for transported ion species. Membrane function, then, seems to be the fmal target for cyclic nucleotide action. The major components of adenosine cyclic 3' ,5'-monophosphate (cAMP) metabolism are schematically diagrammed in figure 1. cAMP has been implicated as the cellular mediator of the following hormones: parathyroid hormone (PTH), antidiuretic hormone (ADH), glucagon, calcitonin (CT), thyroid hormones (T3, T4), catecholamines and angiotensin II.
The initial event in the action of the above hormones is the binding of the hormone to a specific receptor located on the outside of the plasma membrane. Experimental evidence suggests that these receptors, at least for those for PTH 1
Supported by a grant from the 'Deutsche Forschungsgemeinschaft' (Os 42/2-5).
Downloaded by: National Univ. of Singapore 137.132.123.69 - 4/1/2017 12:39:22 PM
Steroid hormones (glucocorticoids and mineralocorticoids) have a different mode of action and will not be discussed here. These lipophilic hormones penetrate through the plasma membrane and elicit their effects primarily on intracellular systems involved with DNA, RNA and protein synthesis.
Renal Adenylate Cyclase Systems
75
HORMONE MEMBRANE
BLOOD MODULATORS ATP
Kinase
ATP Prolein
----''----j..
Proteln- P
Water and Ion permeability
MEMBRANE LUMEN
and ADH, are located on the antiluminal membrane of the various tubular cells. Presumably, the receptors of other hormones will also be found on this antiluminal surface. Once bound to the receptor, the hormonal signal is transmitted to adenylate cyclase via intramembranous elements, known as transducers (1). Although the precise nature of this transduction is unknown, these intramembranous events provide an interesting possibility for regulation of the hormonal signal. The initial intracellular consequence of hormonal action is the conversion of ATP to cAMP. This reaction is catalyzed by the membrane-bound enzyme adenylate cyclase. This enzyme has been extensively studied in the kidney and is known to consist of both a regulatory and a catalytic subunit (2, 3). The structural components of adenylate cyclase appear to be similar for the enzyme found in the cortex and in the medulla. The cellular response to elevated intracellular levels of cAMP is thought to consist in the activation of a family of enzymes which are known as protein kinases. These enzymes catalyze phosphorylation reactions within the cell. In the kidney, the substrates for these enzymes have not been identified. Several different protein kinases have been identified in renal tissue. The cAMP-
Downloaded by: National Univ. of Singapore 137.132.123.69 - 4/1/2017 12:39:22 PM
Fig. 1. General scheme of cAMP-mediated hormonal action on kidney cells. PDE = Cyclic nucleotide phosphodiesterase; Reg., Cat. = regulatory and catalytic subunits of the cAMP-activated protein kinase, respectively; ATP = adenosine triphosphate.
Osswald/N orthru p
76
dependent cytoplasmic protein kinase is activated by the binding of cAMP to the regulatory subunit of the enzyme. This causes dissociation of the enzyme molecule so that the catalytic subunit may phosphorylate its substrate (4-6). Activation of such protein kinase in the kidney has been reported after stimulation by PTH and ADH (1, 7). The final response to hormone action is modification of the permeability characteristics of the plasma membranes to solutes and water. The nature of the link between activation of protein kinase and such permeability changes is unknown.
Localization Since the nephron is composed of several functionally and structurally distinct segments, it is necessary to study hormonal regulation at the different sites along the nephron. The application of micropuncture techniques is limited to the accessible sites of the nephron. Recently developed techniques of tubular microdissection, perfusion of isolated tubules and immunohistochemistry permit one to study sites inaccessible to micropuncture. However, these techniques have become available only recently and knowledge about segmental differences of hormone actions is still limited. The following description of the segmental distribution of adenylate cyclase is based primarily on experimental observations in isolated tubular segments of rnicrodissected rabbit nephrons.
The glomerulus consists of the afferent and efferent arterioles, endothelial and epithelial cells and the Goormaghtigh cells. Small changes in the permeability characteristics of the glomerular capillaries, i.e. hydraulic conductivity (Kf), may have major effects on the rate of fIltration of water and electrolytes. Pertinent to the present review are recent observations that the Kf of rat glomeruli decreases following the administration of agents such as PTH, ADH, angiotensin, prostaglandinS and cAMP (9-13). Sraer et at. (14) and 1m bert et al. (15) have demonstrated that adenylate cyclase can be stimulated in a dosedependent fashion with PTH. Using immunohistochemical methods, Dousa et al. (16) were able to demonstrate and localize cAMP in glomeruli of rats. Following the administration of PTH they found increased cAMP fluorescence, mainly in podocytes and Bowman's capsule. PTH also stimulated the accumulation of cGMP; this occurred predominantly in the cytosol of mesangial cells (fig. 2). Prostaglandins have also been reported to stimulate adenylate cyclase in isolated glomeruli (12). It remains undecided whether these observations have any relevance for glomerular function. i3-Receptors have been reported in the renal vascular bed (3). A close correlation between adenylate cyclase activity and
Downloaded by: National Univ. of Singapore 137.132.123.69 - 4/1/2017 12:39:22 PM
Glomerulus
Renal Adenylate Cyclase Systems
77
Fig. 2. Effects of Pili on immunoreactive cAMP in tubules (a and b) and glomerulus (c and d). a Tubules, control kidney . Note diffuse cytoplasmic staining in some tubules and scanty fluorescence in granules. b Tubules, PTH-perfused kidney. Note diffuse staining in cytoplasm and increase in the number and brightness of granules predominantly but not exclusively at periluminal areas of tubular cells (arrow). c Glomerulus, control kidney. d Glomerulus, PTH-perfused kidney. Note the increase in cytoplasmic fluorescence of podocytes (arrow) and cells in Bowman's space. X 500. With permission of Do usa et aJ. (7).
The Proximal Tubules Phosphate is predominantly reabsorbed in the proximal tubule (17). The first indication of cAMP involvement in the renal action of PTH was the observation by Chase and Aurbach (18) that a marked increase in urinary cAMP excretion preceded phosphaturia in response to PTH. In membrane preparations of the kidney, PTH-sensitive adenylate cyclase has been found by several investigators (4, 19- 21). Charbardes et al. (22) found the highest sensitivity of
Downloaded by: National Univ. of Singapore 137.132.123.69 - 4/1/2017 12:39:22 PM
density of /3-receptors has been described for several tissues, e.g. the peripheral vasculature (2). Release of renin is stimulated by /3-adrenergic agonists. In addition, adrenergic innervation has been demonstrated in the juxtaglomerular apparatus (16a). Therefore, one would anticipate that a specific adenyl ate cyclase may be present in some cell population of the juxtaglomerular apparatus.
Osswald/N orthru p
78
PTX rots
Intact rots
o() 12 2 } ml/h
30
•
:
Renal Adenylate Cyclase Systems H Osswald! and T.E. Northrup Abteilung Pharmakologie der Medizinischen Fakultat der RWfH Aachen, Aachen, and Department of Physiology and Biophysics and Division of Nephrology, Mayo Clinic and Mayo Foundation, Rochester, Minn.
Introduction Transepithelial movement of electrolytes and water is accomplished by controlling both the energy-consuming active transport steps and the permeability characteristics of the membranes across which transport is occurring. Energy for renal transport is basically derived from the hydrolysis of adenosine triphosphate (ATP). In the kidney this reaction is catalyzed by a variety of ATPases, predominantly Na-K-ATPase. There is no evidence that cyclic nucleotides are involved in regulating the activity of Na-K-ATPase in the kidney. Rather, cyclic nucleotides seem to be directly involved in regulating the transport characteristics of membranes in the nephron. These characteristics may involve permeability components or structures associated with various carriers for transported ion species. Membrane function, then, seems to be the fmal target for cyclic nucleotide action. The major components of adenosine cyclic 3' ,5'-monophosphate (cAMP) metabolism are schematically diagrammed in figure 1. cAMP has been implicated as the cellular mediator of the following hormones: parathyroid hormone (PTH), antidiuretic hormone (ADH), glucagon, calcitonin (CT), thyroid hormones (T3, T4), catecholamines and angiotensin II.
The initial event in the action of the above hormones is the binding of the hormone to a specific receptor located on the outside of the plasma membrane. Experimental evidence suggests that these receptors, at least for those for PTH 1
Supported by a grant from the 'Deutsche Forschungsgemeinschaft' (Os 42/2-5).
Downloaded by: National Univ. of Singapore 137.132.123.69 - 4/1/2017 12:39:22 PM
Steroid hormones (glucocorticoids and mineralocorticoids) have a different mode of action and will not be discussed here. These lipophilic hormones penetrate through the plasma membrane and elicit their effects primarily on intracellular systems involved with DNA, RNA and protein synthesis.
Renal Adenylate Cyclase Systems
75
HORMONE MEMBRANE
BLOOD MODULATORS ATP
Kinase
ATP Prolein
----''----j..
Proteln- P
Water and Ion permeability
MEMBRANE LUMEN
and ADH, are located on the antiluminal membrane of the various tubular cells. Presumably, the receptors of other hormones will also be found on this antiluminal surface. Once bound to the receptor, the hormonal signal is transmitted to adenylate cyclase via intramembranous elements, known as transducers (1). Although the precise nature of this transduction is unknown, these intramembranous events provide an interesting possibility for regulation of the hormonal signal. The initial intracellular consequence of hormonal action is the conversion of ATP to cAMP. This reaction is catalyzed by the membrane-bound enzyme adenylate cyclase. This enzyme has been extensively studied in the kidney and is known to consist of both a regulatory and a catalytic subunit (2, 3). The structural components of adenylate cyclase appear to be similar for the enzyme found in the cortex and in the medulla. The cellular response to elevated intracellular levels of cAMP is thought to consist in the activation of a family of enzymes which are known as protein kinases. These enzymes catalyze phosphorylation reactions within the cell. In the kidney, the substrates for these enzymes have not been identified. Several different protein kinases have been identified in renal tissue. The cAMP-
Downloaded by: National Univ. of Singapore 137.132.123.69 - 4/1/2017 12:39:22 PM
Fig. 1. General scheme of cAMP-mediated hormonal action on kidney cells. PDE = Cyclic nucleotide phosphodiesterase; Reg., Cat. = regulatory and catalytic subunits of the cAMP-activated protein kinase, respectively; ATP = adenosine triphosphate.
Osswald/N orthru p
76
dependent cytoplasmic protein kinase is activated by the binding of cAMP to the regulatory subunit of the enzyme. This causes dissociation of the enzyme molecule so that the catalytic subunit may phosphorylate its substrate (4-6). Activation of such protein kinase in the kidney has been reported after stimulation by PTH and ADH (1, 7). The final response to hormone action is modification of the permeability characteristics of the plasma membranes to solutes and water. The nature of the link between activation of protein kinase and such permeability changes is unknown.
Localization Since the nephron is composed of several functionally and structurally distinct segments, it is necessary to study hormonal regulation at the different sites along the nephron. The application of micropuncture techniques is limited to the accessible sites of the nephron. Recently developed techniques of tubular microdissection, perfusion of isolated tubules and immunohistochemistry permit one to study sites inaccessible to micropuncture. However, these techniques have become available only recently and knowledge about segmental differences of hormone actions is still limited. The following description of the segmental distribution of adenylate cyclase is based primarily on experimental observations in isolated tubular segments of rnicrodissected rabbit nephrons.
The glomerulus consists of the afferent and efferent arterioles, endothelial and epithelial cells and the Goormaghtigh cells. Small changes in the permeability characteristics of the glomerular capillaries, i.e. hydraulic conductivity (Kf), may have major effects on the rate of fIltration of water and electrolytes. Pertinent to the present review are recent observations that the Kf of rat glomeruli decreases following the administration of agents such as PTH, ADH, angiotensin, prostaglandinS and cAMP (9-13). Sraer et at. (14) and 1m bert et al. (15) have demonstrated that adenylate cyclase can be stimulated in a dosedependent fashion with PTH. Using immunohistochemical methods, Dousa et al. (16) were able to demonstrate and localize cAMP in glomeruli of rats. Following the administration of PTH they found increased cAMP fluorescence, mainly in podocytes and Bowman's capsule. PTH also stimulated the accumulation of cGMP; this occurred predominantly in the cytosol of mesangial cells (fig. 2). Prostaglandins have also been reported to stimulate adenylate cyclase in isolated glomeruli (12). It remains undecided whether these observations have any relevance for glomerular function. i3-Receptors have been reported in the renal vascular bed (3). A close correlation between adenylate cyclase activity and
Downloaded by: National Univ. of Singapore 137.132.123.69 - 4/1/2017 12:39:22 PM
Glomerulus
Renal Adenylate Cyclase Systems
77
Fig. 2. Effects of Pili on immunoreactive cAMP in tubules (a and b) and glomerulus (c and d). a Tubules, control kidney . Note diffuse cytoplasmic staining in some tubules and scanty fluorescence in granules. b Tubules, PTH-perfused kidney. Note diffuse staining in cytoplasm and increase in the number and brightness of granules predominantly but not exclusively at periluminal areas of tubular cells (arrow). c Glomerulus, control kidney. d Glomerulus, PTH-perfused kidney. Note the increase in cytoplasmic fluorescence of podocytes (arrow) and cells in Bowman's space. X 500. With permission of Do usa et aJ. (7).
The Proximal Tubules Phosphate is predominantly reabsorbed in the proximal tubule (17). The first indication of cAMP involvement in the renal action of PTH was the observation by Chase and Aurbach (18) that a marked increase in urinary cAMP excretion preceded phosphaturia in response to PTH. In membrane preparations of the kidney, PTH-sensitive adenylate cyclase has been found by several investigators (4, 19- 21). Charbardes et al. (22) found the highest sensitivity of
Downloaded by: National Univ. of Singapore 137.132.123.69 - 4/1/2017 12:39:22 PM
density of /3-receptors has been described for several tissues, e.g. the peripheral vasculature (2). Release of renin is stimulated by /3-adrenergic agonists. In addition, adrenergic innervation has been demonstrated in the juxtaglomerular apparatus (16a). Therefore, one would anticipate that a specific adenyl ate cyclase may be present in some cell population of the juxtaglomerular apparatus.
Osswald/N orthru p
78
PTX rots
Intact rots
o() 12 2 } ml/h
30
•
:
