Life Sciences, Vol. Printed in the USA
51, pp. 1699-1703
Pergamon Press
SYNTHESIS AND RELEASE OF ACETYLCHOLINE IN THE RABBIT KIDNEY CORTEX Suzanne Evans, Lal C. Garg and Edwin M. Meyer* Department of Pharmacology and Therapeutics University of Florida College of Medicine Gainesville, Florida 32610 (Received in final form September 21, 1992)
Summary. Several cholinergic processes were demonstrated and partially characterized in rabbit kidney cortical minces: choline uptake, acetylcholine synthesis and calciumdependent release. Minces took up labelled choline, acetylated it, and stored it in a pool that was not readily accessible to physostigmine-sensitive cholinesterase activity. [3H]Acetylcholine synthesis but not [3H]choline uptake was inhibited by the removal of sodium ions or incubation at 0°C. The release of newly synthesized [3H]acetylcholine was increased by 300 mOsmol urea in a calcium-dependent manner, but not by potassium depolarization (300 mOsmol), vasopressin (10 ~tM), or bradykinin (10 p.M). These results suggest that acetylcholine may be synthesized by non-neuronal rabbit kidney cortical ceils and that this transmitter may be released in response to physiological levels of urea. Neural control of renal function is generally considered to be effected by the sympathetic nervous system alone, there being sparse evidence for cholinergic innervation to the kidney (1). However, it is well established that the infusion of acetylcholine (ACh) into the renal blood supply causes diuresis and increases renal blood flow (2-6). These effects are blocked by atropine, suggesting that they are mediated by muscarinic receptors (6). The presence of muscarinic receptors on inner medullary collecting cells was demonstrated recently by McArdle et al. (7) in the rabbit, who also showed that cholinergic agents stimulate phosphoinositide hydrolysis in these cells (8). While there is strong evidence for muscarinic receptors in the kidney (9), the potential source of ACh in this organ is less clear. In the dog, Pirola et al. (10) reported high affinity choline uptake, ACh synthesis, and choline acetyltransferase (CAT) activity in kidney tissues from which neurons of passage were removed. These data supported the concept of a "local" cholinergic system that may modulate tubular reabsorption of sodium and water. However, their results can also be explained if ACh is synthesized in renal epithelial cells. In addition, such local renal cholinergic activity has not been demonstrated in other species such as the rabbit in which renal muscarinic receptor mediated responses are well characterized. Further, it is not known which conditions, if any, trigger the release of renal ACh. The purpose of this study, therefore, was to investigate not only whether ACh is synthesized from choline in another species besides the dog, but to elucidate one or more factors that may be able to modulate its release in this tissue. Materials and Methods Chemicals: All non-radioactive chemicals were purchased from Sigma Chemical Co. (St. louis, MO). [3H-methyl]choline (80 Ci/mM) was purchased from New England Nuclear (Boston, MA). Krebs-Ringer (KR) buffer contained NaC1 (139 mM), KC1 (5.5 raM), CaC12 (1 mM), MgC12 * Corresponding Author: Edwin M. Meyer, Ph.D., Associate Professor, Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Box 100267, Health Science Center, Gainesville, FL 32610-0267. 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
1700
Renal Cholinergic Properties
Vol. 51, No. 22, 1992
(I mM), glucose (10 mM), NaH2PO4 (1 raM), and NaHCO 3 (15 mM). For sodium-free KR buffer, sodium salts were replaced isotonically with Tris-HCl (11 raM) and LiC1 (140 mM). Both solutions were maintained at pH 7.4. All KR buffers were oxygenated with 95/5, O2/CO2,for at least 5 minutes before use. Tissue preparation: Rabbits were decapitated and their kidneys removed. The cortices were quickly dissected away and placed immediately in KR buffer for preparation of minces. Minces were prepared using a Mcllwain tissue chopper, and were suspended in ice-cold, oxygenated KR buffer (setting #4). In one experiment, minces were homogenized in ice-cold water using a glass mortar and teflon pestle (4 passes at a 0.25 mm clearance), centrifuged at 20,000 g for 15 min, and then resuspended in KR buffer for the ACh-synthesis incubation. Measurement of [3H]choline uptake and [3H]ACh synthesis: Minces were added to prewarmed tubes containing a tracer amount of [3H]choline (5.1 Ci/mM), 1 or 20 gM unlabelled choline, and specified drugs, tbr a final incubation volume of 200 gL. Mince incubations were carried out at 37°C for specified intervals (5 or 8 rain). Physostigmine (10 gM) was added to the uptake incubation only when specified. Immediately following incubation, samples were placed on ice and pelleted by centrifugation at 25,000 g for 5 minutes. Pellets were washed and homogenized in water containing 10 ~tM physostigmine. [3H]ACh was separated from [3H]choline by ion pair extraction following phosphorylation of the choline by choline kinase as described previously (11). Aliquots of the organic phase (ACh) and the aqueous phase (phosphorylated choline) were then used for liquid scintillation spectrophotometry, using Liquiscint (National Diagnostics, Ltd.). In some samples treated with choline kinase, sodium tetraphenylboron was removed from the organic phase during the ion-pair extraction (butyronitrile) in order to demonstrate that the radioactive species extracted into that phase was labeled ACh. Measurement of [3H]ACh release: Minces were pre-loaded with a tracer amount of [3H]choline (5.1 Ci/mMol) and 20 gM unlabelled choline. Uptake of the choline was terminated after 10 minutes at 37°C. The tissue was then washed by centrifugation at 12,000 g for 5 minutes, and the supernatant removed. The remaining pellet was resuspended in KR buffer containing 10 gM physostigmine. Aliquots of tissue were then added to pre-warmed tubes containing KR buffer and specified drugs for a 10 min incubation at 37°C (10). Release was stopped by the rapid centrifugation of each sample at 12,000 g for 5 minutes at 4°C. ACh and choline were separated and assayed in the supernatant and tissue as described above. Protein assay: Protein levels were estimated using absorbance at 280 nM in a Beckman DU-7 spectrophotometer. Statistical analyses: Differences among mean values were determined with either the Student's t-test (for comparisons between two means, each used once) or one-way analysis of variance (for simultaneous comparisons of 3 or more means). The latter test used the Statview program on a Maclntosh SE computer. All values are expressed as the means + S.E.M., and each experiment was performed at least three times. Results Minces of rabbit kidney cortex took up 1 or 20 gM [3H]choline in a manner that was unaffected by removal of sodium ions or incubation of the tissue at 0°C (Fig 1A). [3H]ACh was also synthesized in each preparation, and this synthesis was partially dependent on the presence of sodium ions (Fig. 1B). [3H]ACh synthesis and [3H]choline uptake were also observed in minces homogenized in ice-cold water to an extent similar to that seen in intact minces (Fig. 2A). This would be expected if choline uptake and synthesis were occurring within renal tubular cells rather than neurons since the tubular epithelial cells are not susceptible to lysis by hypotonicity, as are nerve endings. Newly synthesized [3H]ACh appeared to be sequestered away from cholinesterase activity in the mince preparation, since addition of 10 t-tM physostigmine to the KR buffer during the uptake incubation had no effect on the levels of the transmitter (mean + S.E.M. [3H]ACh levels in the presence or absence of physostigmine were 0.13 + 0.014 pmol/mg protein and 0.12 + 0.016 pmol/mg protein, respectively).
Vol. 51, No. 22, 1992
'E" T,
Renal Cholinergic Properties
1701
.~
2
A
a.
B
o *
[]
0.4 ~
KR buffer Sodium-free KR buffer, 0°
E
0.2 ¢~
r-
0
0
0.0 1
20
[3H]Choline,
1
p.M
20
[3H]Choline,
'~
p.M
Fig. 1 The uptake of [3H]choline (A) and [3HIACh synthesis (B) in rabbit kidney cortex minces. [3H]Choline (1 or 20 ~M) was incubated with the tissues for 8 minutes at 0°C or 37°C in KR buffer, with or without sodium ions, as described in the text. The total uptake of [3H]choline was measured after washing the tissue. [3H]ACh levels were assayed as described in the text. Each.value is the mean + S.E.M. of 4 animals, with each assay performed in duplicate. *p < 0.05 compared to control value, one-way analysis of variance. Ac-
0.2
o
IB
o
e~ 13
*
[]
+Calcium
o E n
t- 40
-3o
~.
• 20
~
"10
I~ t-
o
0.1 o
>
i
o o Q
iO.G
Mince
Homogenate
-6
Preparation
~+
~
._c
._c
E o
~
,~
c~
E
> o
Fig. 2 [3H]Choline uptake and [3H]ACh synthesis and release of newly synthesized [3H]ACh from rabbit kidney_ cortex. A. Minces and their homogenates were incubated for 5 min at 37°C with 1 ~M [3H]choline and then assayed for [3H]cboline uptake and [3H]ACh synthes,.'s as described in the text. Each value is the mean + S.E.M. of at least 3 tissue preparations/group, each preparation assayed in duplicate. B. Cortical minces preincubated with [3H]choline to load them with [3H]ACh as described in the text were incubated for 10 min at 37°C in the presence of 150 mM KC1, 300 mM urea, 10 ~VI vasopressin, or 10 IxM bradykinin. Release is shown both in the presence and absence of extracellular calcium. Values are expressed as a percentage of the total [3H]ACh in the tissue. N = 3 animals per group, each measured in duplicate. *p < 0.05 compared to control group, same calcium concentration, one-way analysis of variance.
0
1702
Renal Cholinergic Properties
Vol. 51, No. 22, 1992
The release of newly synthesized [3H]ACh from minces of kidney cortex was stimulated by the presence of urea (300 mOsmol) in the KR buffer (Fig. 2B). This release was dependent on calcium. Release was not affected by the addition to the KR buffer of several other agents: K+ (300 mOsmol), vasopressin (10 gM), or bradykinin (10 gM) (Fig. 2B). Discussion Much of our knowledge about the synthesis and release of ACh derives from nervous tissue. Neuronal ACh is synthesized preferentially from choline taken up by a sodium-dependent transport process (12-14). In contrast, sodium-independent choline uptake is found associated with most cell types and is not coupled to ACh synthesis. Newly synthesized neuronal ACh is released in response to depolarization-induced calcium influx by a mechanism that remains obscure. High affinity choline uptake was recently observed in dog kidney tissue and it was accordingly hypothesized that this uptake may be associated with intrinsic cholinergic nerve terminals (10). However, the calcium-dependent release of ACh was not reported, leaving unclear a functional role for the transmitter in this tissue. Our results demonstrate for the first time the synthesis, physostigmine-independent storage, and calcium-dependent release of ACh in the rabbit kidney cortex. Most or all of the [3H]choline uptake by kidney cortical minces appeared to be by a sodiumindependent process, even at the lower, 1 ~M concentration. This low concentration of choline was used because it approximates the Kin value for sodium dependent, high affinity transport in cholinergic neurons, thus optimizing the likelihood of observing such sodium-dependent uptake. In contrast, the high choline concentration was about 10 times the Kin value for high affinity choline uptake, and it was expected to result in a larger fraction of low versus high affinity choline uptake. Despite the present negative results with respect to sodium-dependent uptake at both concentrations, it remains possible that this type of uptake is present but masked by much larger rates of low affinity uptake. Consistent with this masking possibility is the observation that most of the ACh synthesis in the tissue minces is sensitive to sodium ion removal or incubation at 0°C. While this argues in favor of there being a high affinity uptake of choline that is coupled to ACh synthesis, two observations argue against any conclusion that the ACh synthesis is neuronal in location. First, the resistance of ACh synthesis to a hypo-osmotic shock during homogenization is not consistent with a nerve terminal location, since isolated nerve terminals usually lyse under this condition. Further, depolarization with potassium ions did not release the newly synthesized ACh in a calcium-dependent manner as it does in neuronally derived preparations. Regardless of whether ACh is localized in neurons or another type of cell in the kidney, it must be stored in a pool sequestered from cholinesterase activity and available for release under appropriate conditions to have any modulatory role in the kidney. The absence of any protective action of physostigmine on ACh levels suggests that the newly synthesized transmitter is already sequestered against hydrolysis by this enzyme. With respect to release, only 300 mOsmol urea of the various potential secretagogues tested triggered the calcium-dependent release of ACh. Addition of potassium ions to the same osmolarity did not increase release over control levels, which suggests that the effect was due to the urea per se rather than the osmolarity. The apparent secretagogue effect of urea is an interesting one that merits further investigation to elucidate a possible physiological role for ACh. We were able to demonstrate 300 mM ureainduced release of [3H]ACh from rat neocortical P2 fractions after similar incubations at 37°C, further suggesting that this observation in kidney is non-neuronal in origin (J. Hicks and E. Meyer, unpublished observation). Therefore, it is possible to speculate that an increase in the urea concentration in the tubule cells might trigger a release of ACh, the effect of which would be diuresis. ACh in the kidney may therefore play a role which is more modulatory in nature than its neurotransmitter role. Garg et al. (15) have shown that increasing the osmolarity decreases the carbachol-stimulated breakdown of phosphoinositides. A link between the concentration of urea and the action of ACh would therefore appear feasible.
Vol. 51, No. 22, 1992
Renal Cholinergic Properties
1703
ACh synthesized locally within the kidney may act as autocrine or paracrine substance producing an increase in sodium excretion as is the case with dopamine (16, 17). Dopamine is synthesized in the kidney from L-dopa (16) and dopamine receptors are present in the kidney (17). Intrarenal dopamine has been shown to produce sodium excretion under conditions of moderate sodium loading (18). Exogeneous administration of ACh also produces natriuresis (6) and muscarinic receptors are present in the kidney (7, 9). It is possible that multiple control mechanisms are involved in sodium excretion during sodium loading and locally produced ACh may also play a role in sodium excretion under certain conditions. However, this remains to be determined. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
V.H.GATTONE, C.F. MARFURT and S. DALLIE. Am. J. Physiol. 250 F189-F196 (1986). S.Z. FADEM, G. HERNANDEZ-LLAMAS, R.V. PATAK, S.G. ROSENBLATT, M.D. LIFSCHITZ and J.H. STEIN. J. Clin. Invest. 69 604-610 (1982). D.A. HARTUPEE, J.D. BURNETT, JR., J.I. MERTZ and F.G. KNOX. Am. J. Physiol. 243 F325-F329 (1982). J.P. HAYSLETT, D.T. DAMATO, M. KASHGARIAN and F.H. EPSTEIN. Am. J. Physiol. 218 880-885 (1970). N. LAMEIRE, R. VANHOLDER, S. RINGOIR and L. LEUSEN. Circ. Res. 47 839-844 (1980). A.J. VANDER. Am. J. Physiol. 20__fi6492-498 (1964). S. MCARDLE, L.C. GARG and F.T. CREWS. J. Pharm. Exp. Ther. 248 12-16 (1989). S. MCARDLE and L.C. GARG. J. Pharm. Exp. Ther. 248 682-686 (1989). L.C. GARG. Pharmacol. Rev. 4J 81-102 (1992). C.J. PIROLA, A.L. ALVAREZ, M.S. BALDA, S. FINKIELMAN and V.E. NAHMOD. Am. J. Physiol. 257 F746-F754 (1989). E.M. MEYER and D.H. OTERO. J. Neurosci. 5_ 1202-1207 (1985). R.S. JOPE. Brain Res. Rev. 1 313-344 (1979). E.M. MEYER, D.G. ENGLE and J.R. COOPER. Neurochem. Res. 7 749-759 (1982). H.I. YAMAMURA and S.H. SNYDER. J. Neurochem. 21 1355-1374 (1973). L.C. GARG, E. KAPTURCZAK and S. MCARDLE. J. Pharm. Exp. Ther. 247 495-501 (1988). I. SERI, B.C. KONE, S.R. GULLANS, A. APERIA, B.M. BRENNER and B.J. BALLERMANN. Am. J. Physiol. 255 F666-F673 (1988). H.M. SIRAGY, R.A. FELDER, N.L. HOWELL, R.L. CHEVALIER, M.J. PEACH and R.M. CAREY. Am. J. Physiol. 257 F469-F477 (1989).
51, pp. 1699-1703
Pergamon Press
SYNTHESIS AND RELEASE OF ACETYLCHOLINE IN THE RABBIT KIDNEY CORTEX Suzanne Evans, Lal C. Garg and Edwin M. Meyer* Department of Pharmacology and Therapeutics University of Florida College of Medicine Gainesville, Florida 32610 (Received in final form September 21, 1992)
Summary. Several cholinergic processes were demonstrated and partially characterized in rabbit kidney cortical minces: choline uptake, acetylcholine synthesis and calciumdependent release. Minces took up labelled choline, acetylated it, and stored it in a pool that was not readily accessible to physostigmine-sensitive cholinesterase activity. [3H]Acetylcholine synthesis but not [3H]choline uptake was inhibited by the removal of sodium ions or incubation at 0°C. The release of newly synthesized [3H]acetylcholine was increased by 300 mOsmol urea in a calcium-dependent manner, but not by potassium depolarization (300 mOsmol), vasopressin (10 ~tM), or bradykinin (10 p.M). These results suggest that acetylcholine may be synthesized by non-neuronal rabbit kidney cortical ceils and that this transmitter may be released in response to physiological levels of urea. Neural control of renal function is generally considered to be effected by the sympathetic nervous system alone, there being sparse evidence for cholinergic innervation to the kidney (1). However, it is well established that the infusion of acetylcholine (ACh) into the renal blood supply causes diuresis and increases renal blood flow (2-6). These effects are blocked by atropine, suggesting that they are mediated by muscarinic receptors (6). The presence of muscarinic receptors on inner medullary collecting cells was demonstrated recently by McArdle et al. (7) in the rabbit, who also showed that cholinergic agents stimulate phosphoinositide hydrolysis in these cells (8). While there is strong evidence for muscarinic receptors in the kidney (9), the potential source of ACh in this organ is less clear. In the dog, Pirola et al. (10) reported high affinity choline uptake, ACh synthesis, and choline acetyltransferase (CAT) activity in kidney tissues from which neurons of passage were removed. These data supported the concept of a "local" cholinergic system that may modulate tubular reabsorption of sodium and water. However, their results can also be explained if ACh is synthesized in renal epithelial cells. In addition, such local renal cholinergic activity has not been demonstrated in other species such as the rabbit in which renal muscarinic receptor mediated responses are well characterized. Further, it is not known which conditions, if any, trigger the release of renal ACh. The purpose of this study, therefore, was to investigate not only whether ACh is synthesized from choline in another species besides the dog, but to elucidate one or more factors that may be able to modulate its release in this tissue. Materials and Methods Chemicals: All non-radioactive chemicals were purchased from Sigma Chemical Co. (St. louis, MO). [3H-methyl]choline (80 Ci/mM) was purchased from New England Nuclear (Boston, MA). Krebs-Ringer (KR) buffer contained NaC1 (139 mM), KC1 (5.5 raM), CaC12 (1 mM), MgC12 * Corresponding Author: Edwin M. Meyer, Ph.D., Associate Professor, Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Box 100267, Health Science Center, Gainesville, FL 32610-0267. 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
1700
Renal Cholinergic Properties
Vol. 51, No. 22, 1992
(I mM), glucose (10 mM), NaH2PO4 (1 raM), and NaHCO 3 (15 mM). For sodium-free KR buffer, sodium salts were replaced isotonically with Tris-HCl (11 raM) and LiC1 (140 mM). Both solutions were maintained at pH 7.4. All KR buffers were oxygenated with 95/5, O2/CO2,for at least 5 minutes before use. Tissue preparation: Rabbits were decapitated and their kidneys removed. The cortices were quickly dissected away and placed immediately in KR buffer for preparation of minces. Minces were prepared using a Mcllwain tissue chopper, and were suspended in ice-cold, oxygenated KR buffer (setting #4). In one experiment, minces were homogenized in ice-cold water using a glass mortar and teflon pestle (4 passes at a 0.25 mm clearance), centrifuged at 20,000 g for 15 min, and then resuspended in KR buffer for the ACh-synthesis incubation. Measurement of [3H]choline uptake and [3H]ACh synthesis: Minces were added to prewarmed tubes containing a tracer amount of [3H]choline (5.1 Ci/mM), 1 or 20 gM unlabelled choline, and specified drugs, tbr a final incubation volume of 200 gL. Mince incubations were carried out at 37°C for specified intervals (5 or 8 rain). Physostigmine (10 gM) was added to the uptake incubation only when specified. Immediately following incubation, samples were placed on ice and pelleted by centrifugation at 25,000 g for 5 minutes. Pellets were washed and homogenized in water containing 10 ~tM physostigmine. [3H]ACh was separated from [3H]choline by ion pair extraction following phosphorylation of the choline by choline kinase as described previously (11). Aliquots of the organic phase (ACh) and the aqueous phase (phosphorylated choline) were then used for liquid scintillation spectrophotometry, using Liquiscint (National Diagnostics, Ltd.). In some samples treated with choline kinase, sodium tetraphenylboron was removed from the organic phase during the ion-pair extraction (butyronitrile) in order to demonstrate that the radioactive species extracted into that phase was labeled ACh. Measurement of [3H]ACh release: Minces were pre-loaded with a tracer amount of [3H]choline (5.1 Ci/mMol) and 20 gM unlabelled choline. Uptake of the choline was terminated after 10 minutes at 37°C. The tissue was then washed by centrifugation at 12,000 g for 5 minutes, and the supernatant removed. The remaining pellet was resuspended in KR buffer containing 10 gM physostigmine. Aliquots of tissue were then added to pre-warmed tubes containing KR buffer and specified drugs for a 10 min incubation at 37°C (10). Release was stopped by the rapid centrifugation of each sample at 12,000 g for 5 minutes at 4°C. ACh and choline were separated and assayed in the supernatant and tissue as described above. Protein assay: Protein levels were estimated using absorbance at 280 nM in a Beckman DU-7 spectrophotometer. Statistical analyses: Differences among mean values were determined with either the Student's t-test (for comparisons between two means, each used once) or one-way analysis of variance (for simultaneous comparisons of 3 or more means). The latter test used the Statview program on a Maclntosh SE computer. All values are expressed as the means + S.E.M., and each experiment was performed at least three times. Results Minces of rabbit kidney cortex took up 1 or 20 gM [3H]choline in a manner that was unaffected by removal of sodium ions or incubation of the tissue at 0°C (Fig 1A). [3H]ACh was also synthesized in each preparation, and this synthesis was partially dependent on the presence of sodium ions (Fig. 1B). [3H]ACh synthesis and [3H]choline uptake were also observed in minces homogenized in ice-cold water to an extent similar to that seen in intact minces (Fig. 2A). This would be expected if choline uptake and synthesis were occurring within renal tubular cells rather than neurons since the tubular epithelial cells are not susceptible to lysis by hypotonicity, as are nerve endings. Newly synthesized [3H]ACh appeared to be sequestered away from cholinesterase activity in the mince preparation, since addition of 10 t-tM physostigmine to the KR buffer during the uptake incubation had no effect on the levels of the transmitter (mean + S.E.M. [3H]ACh levels in the presence or absence of physostigmine were 0.13 + 0.014 pmol/mg protein and 0.12 + 0.016 pmol/mg protein, respectively).
Vol. 51, No. 22, 1992
'E" T,
Renal Cholinergic Properties
1701
.~
2
A
a.
B
o *
[]
0.4 ~
KR buffer Sodium-free KR buffer, 0°
E
0.2 ¢~
r-
0
0
0.0 1
20
[3H]Choline,
1
p.M
20
[3H]Choline,
'~
p.M
Fig. 1 The uptake of [3H]choline (A) and [3HIACh synthesis (B) in rabbit kidney cortex minces. [3H]Choline (1 or 20 ~M) was incubated with the tissues for 8 minutes at 0°C or 37°C in KR buffer, with or without sodium ions, as described in the text. The total uptake of [3H]choline was measured after washing the tissue. [3H]ACh levels were assayed as described in the text. Each.value is the mean + S.E.M. of 4 animals, with each assay performed in duplicate. *p < 0.05 compared to control value, one-way analysis of variance. Ac-
0.2
o
IB
o
e~ 13
*
[]
+Calcium
o E n
t- 40
-3o
~.
• 20
~
"10
I~ t-
o
0.1 o
>
i
o o Q
iO.G
Mince
Homogenate
-6
Preparation
~+
~
._c
._c
E o
~
,~
c~
E
> o
Fig. 2 [3H]Choline uptake and [3H]ACh synthesis and release of newly synthesized [3H]ACh from rabbit kidney_ cortex. A. Minces and their homogenates were incubated for 5 min at 37°C with 1 ~M [3H]choline and then assayed for [3H]cboline uptake and [3H]ACh synthes,.'s as described in the text. Each value is the mean + S.E.M. of at least 3 tissue preparations/group, each preparation assayed in duplicate. B. Cortical minces preincubated with [3H]choline to load them with [3H]ACh as described in the text were incubated for 10 min at 37°C in the presence of 150 mM KC1, 300 mM urea, 10 ~VI vasopressin, or 10 IxM bradykinin. Release is shown both in the presence and absence of extracellular calcium. Values are expressed as a percentage of the total [3H]ACh in the tissue. N = 3 animals per group, each measured in duplicate. *p < 0.05 compared to control group, same calcium concentration, one-way analysis of variance.
0
1702
Renal Cholinergic Properties
Vol. 51, No. 22, 1992
The release of newly synthesized [3H]ACh from minces of kidney cortex was stimulated by the presence of urea (300 mOsmol) in the KR buffer (Fig. 2B). This release was dependent on calcium. Release was not affected by the addition to the KR buffer of several other agents: K+ (300 mOsmol), vasopressin (10 gM), or bradykinin (10 gM) (Fig. 2B). Discussion Much of our knowledge about the synthesis and release of ACh derives from nervous tissue. Neuronal ACh is synthesized preferentially from choline taken up by a sodium-dependent transport process (12-14). In contrast, sodium-independent choline uptake is found associated with most cell types and is not coupled to ACh synthesis. Newly synthesized neuronal ACh is released in response to depolarization-induced calcium influx by a mechanism that remains obscure. High affinity choline uptake was recently observed in dog kidney tissue and it was accordingly hypothesized that this uptake may be associated with intrinsic cholinergic nerve terminals (10). However, the calcium-dependent release of ACh was not reported, leaving unclear a functional role for the transmitter in this tissue. Our results demonstrate for the first time the synthesis, physostigmine-independent storage, and calcium-dependent release of ACh in the rabbit kidney cortex. Most or all of the [3H]choline uptake by kidney cortical minces appeared to be by a sodiumindependent process, even at the lower, 1 ~M concentration. This low concentration of choline was used because it approximates the Kin value for sodium dependent, high affinity transport in cholinergic neurons, thus optimizing the likelihood of observing such sodium-dependent uptake. In contrast, the high choline concentration was about 10 times the Kin value for high affinity choline uptake, and it was expected to result in a larger fraction of low versus high affinity choline uptake. Despite the present negative results with respect to sodium-dependent uptake at both concentrations, it remains possible that this type of uptake is present but masked by much larger rates of low affinity uptake. Consistent with this masking possibility is the observation that most of the ACh synthesis in the tissue minces is sensitive to sodium ion removal or incubation at 0°C. While this argues in favor of there being a high affinity uptake of choline that is coupled to ACh synthesis, two observations argue against any conclusion that the ACh synthesis is neuronal in location. First, the resistance of ACh synthesis to a hypo-osmotic shock during homogenization is not consistent with a nerve terminal location, since isolated nerve terminals usually lyse under this condition. Further, depolarization with potassium ions did not release the newly synthesized ACh in a calcium-dependent manner as it does in neuronally derived preparations. Regardless of whether ACh is localized in neurons or another type of cell in the kidney, it must be stored in a pool sequestered from cholinesterase activity and available for release under appropriate conditions to have any modulatory role in the kidney. The absence of any protective action of physostigmine on ACh levels suggests that the newly synthesized transmitter is already sequestered against hydrolysis by this enzyme. With respect to release, only 300 mOsmol urea of the various potential secretagogues tested triggered the calcium-dependent release of ACh. Addition of potassium ions to the same osmolarity did not increase release over control levels, which suggests that the effect was due to the urea per se rather than the osmolarity. The apparent secretagogue effect of urea is an interesting one that merits further investigation to elucidate a possible physiological role for ACh. We were able to demonstrate 300 mM ureainduced release of [3H]ACh from rat neocortical P2 fractions after similar incubations at 37°C, further suggesting that this observation in kidney is non-neuronal in origin (J. Hicks and E. Meyer, unpublished observation). Therefore, it is possible to speculate that an increase in the urea concentration in the tubule cells might trigger a release of ACh, the effect of which would be diuresis. ACh in the kidney may therefore play a role which is more modulatory in nature than its neurotransmitter role. Garg et al. (15) have shown that increasing the osmolarity decreases the carbachol-stimulated breakdown of phosphoinositides. A link between the concentration of urea and the action of ACh would therefore appear feasible.
Vol. 51, No. 22, 1992
Renal Cholinergic Properties
1703
ACh synthesized locally within the kidney may act as autocrine or paracrine substance producing an increase in sodium excretion as is the case with dopamine (16, 17). Dopamine is synthesized in the kidney from L-dopa (16) and dopamine receptors are present in the kidney (17). Intrarenal dopamine has been shown to produce sodium excretion under conditions of moderate sodium loading (18). Exogeneous administration of ACh also produces natriuresis (6) and muscarinic receptors are present in the kidney (7, 9). It is possible that multiple control mechanisms are involved in sodium excretion during sodium loading and locally produced ACh may also play a role in sodium excretion under certain conditions. However, this remains to be determined. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
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