Effects of central and peripheral neuropeptide Y on sodium and water excretion in the rat

Physiology& Behavior, Vol. 46, pp. 9-11. ©Pergamon Press plc, 1989. Printed in the U.S.A.

0031-9384/89 $3.00 + .00

Effects of Central and Peripheral Neuropeptide Y on Sodium and Water Excretion in the Rat D O N A L D D. S M Y T H , *1 J. R O G E R W I L S O N , t E R I C S E I D L I T Z t A N D S H A R O N L. T H O M *

Departments o f *Pharmacology & Therapeutics and "PPsychology University o f Manitoba, Winnipeg, Manitoba, Canada R3E OW3

SMYTH, D. D., J. R. WILSON, E. SEIDLITZ AND S. L. THOM. Effects of central and peripheral neuropeptide Y on sodium and water excretion in the rat. PHYSIOL BEHAV 46(1) 9-11, 1989.--Neuropeptide Y (NPY) is widely distributed throughout the central nervous system and in several sympathetically innervated tissues, including the kidney. Although many central and peripheral acting endogenous compounds alter renal function, the role of NPY is unknown. Accordingly, we examined the effects of intracerebroventricular (ICV) or intrarenal administration of NPY on sodium and water excretion in the barbiturate anesthetized rat. Sprague-Dawley rats were uninephrectomized 10 days prior to testing and, in rats undergoing ICV administration, cannulae were implanted 3 days prior to testing. For testing, rats were anesthetized (Nembutal) and the jugular vein, renal artery and ureter catheterized. The results showed that the intrarenal infusion of NPY at 1 I~g/kg/min increased sodium and water excretion relative to the saline control group without altering blood pressure or creatinine clearance. Similarly, ICV administration of NPY at 10 Izg in a 5 ttl volume increased the excretion of sodium and water without altering blood pressure as compared to the artificial CSF group. These findings suggest that both central and peripheral NPY may contribute to the regulation of sodium and water excretion in the rat. Neuropeptide Y

Renal function

Intracerebroventricular (ICV)

STUDIES have revealed the widespread distribution, and high concentration, of NPY throughout the mammalian central and peripheral nervous system (3, 13, 14, 18). Centrally, NPY immunoreactivity has been associated with catecholamines in brainstem and sympathetic neurons (9,14). The fact that NPYcontaining postganglionic fibers innervate, among other tissues, the kidney (3) suggests a role of NPY in autonomic regulation. As well, the effects of NPY have been closely associated with the effects of catecholamines (19). Other studies report similarities between the effects obtained with NPY and alpha2-adrenoceptor agonists. For example, both NPY and alpha2-adrenoceptor agonists inhibit renin release in the kidney by activation of a pertussis toxin sensitive protein associated with the adenylate cyclase cAMP system (8,15). Moreover, the central administration of NPY or clonidine, an alpha2-adrenoceptor agonist, tends to decrease blood pressure (6, 10, 12) and attenuate forskolin-stimulated cAMP production in feline cerebral arteries (5). Central and renal alpha2-adrenoceptors also participate in the regulation of fluid and electrolyte balance. For example, natduresis and diuresis are evoked by either central (12) or intrarenal (16,17) administration of clonidine. Nevertheless, the effects of central administration of NPY on renal function is unknown.

Intrarenal

Anesthetized

Thus, the present study examined the effects of NPY administration, by either intracerebroventricular (ICV) infusion or by direct infusion in the renal artery, on the excretion of sodium and water. METHOD

Experimental Pretreatment Male Sprague-Dawley rats weighing 250 to 300 g were unilaterally nephrectomized 7 to 10 days prior to the experiment under ether anesthesia. Chronic ICV cannulae were implanted (see below) at least 3 days prior to the experiment in the rats receiving central NPY (Peninsula Laboratories Inc.) administration. Animals were allowed free access to water (tap water) and chow (Purina Rat Chow) and housed at 230(2 with a 12/12 hr light/dark cycle.

Intracerebroventricular Cannulae A cannulae was implanted into the right lateral cerebral ventricle. Rats were anesthetized (Nembutal, BDH) and placed in a stereotaxic apparatus. A small hole was made in the skull with a hand held drill. The cannulae was a modified tuberculin syringe.

1Requests for reprints should be addressed to Donald D. Smyth, Ph.D., Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, 770 Bannatyne Ave., Winnipeg, Manitoba R3E 0W3.

SMYTH ET AL.

10

Briefly, the syringe tip and corresponding needle were threaded firmly together and a 24-gauge guide tube mounted inside the tip with acrylic cement to a depth of 5 mm. A 31-gauge injector tube was made such that when inserted it would extend 1.5 m m below the guide tube. The tip of the guide tube was implanted with the coordinates, R C = 0 m m from bregma, L = l . 7 m m from the midsagittal suture and V = 3.0 m m from the surface of the skull. These coordinates left the tip of the guide tube 1.0 mm above the right lateral ventricle such that when the injector stylet was mounted into the guide tube on the day of the experiment, the ventricle was entered. The cannulae was secured with 3 stainless steel jeweller's screws and cranioplastic cement. A plug was placed into the guide tube until the day of the experiment. Placement of the cannulae was confirmed at the end of the experiment by the injection of l0 Ixl of dye.

Experimental Protocol On the day of the experiment, rats were anesthetized (Nembutal, BDH; 50 mg/kg IP). A tracheostomy was performed and the animal was placed on a respirator if necessary. Body temperature was maintained (38°C) with a small animal heating blanket and a rectal thermometer connected to a Harvard Animal Blanket Control Unit. A polyethylene catheter (PE60) was placed in the left carotid artery and blood pressure recorded with a Statham pressure transducer (Model P23Dc) connected to a Grass polygraph Model V. A catheter was placed in the left jugular vein for the infusion of saline. The left kidney was exposed by a flank incision and the left ureter cannulated (PE50). In the experiments studying the effects of an intrarenal infusion of NPY, a 30-gauge stainless steel needle was inserted into the aorta opposite the renal artery and advanced into the renal artery. A baseline level of sodium and water excretion was established by the infusion of saline at 0.097 ml/min immediately following the completion of surgery (i.e., the start of the 30-min stabilization period). In the experiments studying the effects of ICV administration of NPY, 8 consecutive 10-min urine collections were obtained. Following the second urine collection, 5 ixl of either artificial cerebrospinal fluid (CSF) or NPY (10 Ixg/5 jxl) was administered into the ICV cannulae over a period of 2 rain. The effects of this infusion were studied over the next 6 urine collections. In the next series of experiments, the effect of NPY infused directly into the renal artery was determined. In these experiments, 6 consecutive 15-min urine collections were obtained. The infusion of vehicle (saline) or NPY (1 txg/kg/min) into the renal artery at 0.0034 ml/min was started immediately following the second urine collection and maintained for the duration of the experiment. Plasma and urine sodium and potassium concentrations were determined with a Beckman Klina Flame Photometer. Creatinine concentrations were determined by a modified Jaffe method with a Beckman Creatinine Analyzer Model 2. Urine osmolality was measured with a microOsmette (Precision Systems). Data were analyzed with a 2 × 7 (drug × time) repeated measures analysis of variance. Where the interaction was significant, the locus of the differences were determined by Duncan's multiple comparison. Data are expressed as the mean -+ the standard error of the mean. RESULTS

Intracerebroventricular Administration During the first two control collection periods, no differences were observed between groups. The fifth collection period is representative of differences observed between groups following the experimental interventions and this data has been presented in Table 1. NPY (10 p~g/5 Ixl) administered ICV failed to alter blood

TABLE 1 EFFECT OF NEUROPEPTIDE Y (NPY) ON RENAL SODIUM AND WATER EXCRETION FOLLOWING INTRARENAL AND INTRACEREBROVENTRICULAR (ICV) ADMINISTRATION

Intrarenal (~g/kg/min) Saline BP rnmHg HRbpm Ccr ml/min UVml/min UNaV ixEq/min UKV p~Eq/min UosmmOsm CH20 ixl/min Cosrn pA/min n

121 396 1.8 37 7.3 4.3 937 -70 107

+- 7 ± 15 ± 0.1 ± 7 ± 1.5 --- 0.4 --- 78 ± 8 - 14 7

1 127 400 2.0 81 12.6 5.4 627 -70 151

ICV (ixg/5 ~xl) CSF

10

± 2 134 ± 3 123 - 4 ± 10 404 _ 7 373 ± 15 ± 0.3 1.4 _ 0.1 2.4 ± 0.2* +- 10" 27 ± 8 52 ± 5* ± 1.4" 4.2 __. 1.4 8.9 ± 1.0" ± 0.4 3.1 ± 0.4 5.0 ___ 0.2* ± 99 992 ± 133 733 ± 49 ± 13 - 4 7 ± 5 - 7 2 ± 4* ± 15" 73 ± 13 125 - 4* 9 5 6

Values are the means ± S.E. of data obtained during the sixth (intrarenal) and fifth (ICV) collection periods. CSF, artificial cerebrospinal fluid; BP, blood pressure; HR, heart rate; bpm, beats per minute; Ccr, creatinine clearance; UV, urine volume; UNaV, sodium excretion; UKV, potassium excretion; Uosm, urine osmolality; CH20, free water clearance; Cosm, osmolar clearance. *denotes p<0.05 versus control (CSF or Saline). pressure or heart rate at the dose investigated. An increase in creatinine clearance was observed as compared to control group receiving the artificial CSF (1.4-+0.1 vs. 2.4-+0.2 ml/min). As well, a two-fold increase in urine volume (27-+8 vs. 52-+5 ~l/min) and sodium excretion (4.2 -+ 1.4 vs. 8.9-+ 1.0 txEq/min) was observed. Potassium excretion was also increased (3.1 -+ 0.4 vs. 5.0 -+ 0.2 txEq/min). Osmolar clearance was increased (73 ± 13 vs. 125 -+ 4 pJ/min), whereas free water clearance was decreased ( - 47 -+ 5 vs. - 72 -+ 4 txl/min) following the NPY administration.

lntrarenal Administration The first two control periods failed to demonstrate any differences between the groups studied. However, the sixth collection period is representative of the post treatment differences observed between groups and this has been presented in detail (Table 1). At the infusion rate studied, NPY failed to alter blood pressure, creatinine clearance or heart rate. This dose did, however, increase urine volume (37-+7 vs. 81 -+ 10 I~l/min) and sodium excretion (7.3 -+ 1.5 vs. 12.6 -+ 1.4 p,Eq/min) but not potassium excretion (4.3-+0.4 vs. 5.4-+0.4 ixEq/min) as compared to the saline vehicle group. Urine osmolality was decreased (937---78 vs. 627 + 99 mOsm) by the NPY infusion. Osmolar clearance was increased (107 -+ 14 vs. 151 -+ 15 ~l/min) and free water clearance was unaltered ( - 70 -+ 8 vs. - 70 -+ 13 ~l/min). DISCUSSION

The present study documents, for the first time, the ability of central (ICV) and peripheral (intrarenal) administration of NPY to increase the excretion of sodium and water. It was based on two considerations. First, the paucity of information relating NPY to fluid and electrolyte balance. Second, the prevailing evidence that regardless of the route of administration, NPY exerts an effect on a number of systems comparable to alpha2-adrenoceptor agonists. This evidence would suggest that the natriuretic and diuretic effects of NPY would approximate those obtained for alpha2adrenoceptor agonists. This has been largely confirmed in the present study. The results showed that intrarenal administration of

NPY AND SODIUM EXCRETION

11

NPY enhanced sodium and water excretion. The absence of significant changes in blood pressure heart rate or creatinine clearance suggests that the observed effects were due to a direct tubular action and not secondary to an increased renal perfusion pressure as previously documented (1). However, unlike other systems studied, discrepancies were observed between the renal effects of NPY and those of alpha2-adrenoceptor agonists. Although both NPY (present study) and alpha2-adrenoceptor agonists (7) increase water and sodium excretion, the increase in free water clearance which is characteristic for alpha2-adrenoceptor stimulation was not found with NPY. This indicates that the effects of NPY may not be mediated through the antagonism of vasopressin. Interestingly, a recent study has reported that an infusion rate of NPY comparable to that used in the present study decreased plasma vasopressin levels (2) which would conceivably increase free water clearance. The reason the present study did not observe an increase in free water clearance is unknown. The effect of an ICV infusion of NPY was also determined. Again, the dose chosen failed to alter blood pressure and heart rate which has been observed with higher doses (10). Creatinine clearance was significantly increased. Urine volume, sodium excretion and potassium excretion were significantly increased at

the dose investigated. These results are consistent with that observed for alpha2-adrenoceptor agonists administered into the ICV (12). This previous study proposed that the natriuresis and diuresis observed following the administration of clonidine was secondary to a decrease in the level of sympathetic renal nerve activity. Whether this is the mechanism by which NPY also mediates the natriuresis and diuresis remains to be determined. The increase in glomerular filtration rate, as measured by creatinine clearance, may explain in part the observed renal effects. In summary, the present study has demonstrated a diuresis and natriuresis following the administration of NPY either directly into the renal artery or ICV. The absence of alterations in blood pressure suggest that this is due to a direct renal effect and not secondary to altered cardiovascular parameters. Whether these effects of NPY are coupled to a pertussis toxin sensitive G protein, as has been documented in other tissues (11), remains to be determined. ACKNOWLEDGEMENTS This study was funded by the Medical Research Council of Canada and the Manitoba Heart Foundation. D.D.S. is the recipient of a Canadian Heart Foundation Scholarship (1986-1991).

REFERENCES 1. Allen, J. M.; Raine, A. E. G.; Ledingham, J. G. G.; Bloom, S. R. Neuropeptide Y: a novel renal peptide with vasoconstrictor and natriuretic activity. Clin. Sci. 68:373-377; 1985. 2. Aubert, J. F.; Burnier, M.; Waeber, B.; Nussberger, J.; Dipette, D. J.; Bun'is, J. F.; Brunner, H. Effects of a nonpressor dose of neuropeptide Y on cardiac output, regional blood flow distribution and plasma renin, vasopressin and catecholamine levels. J. Pharmacol. Exp. Ther. 244:1109-1115; 1988. 3. Ballesta, J.; Polak, J. M.; Allen, J. M.; Bloom, S. R. The nerves of the juxtaglomerular apparatus of man and other mammals contain the potent peptide NPY. Histochemistry 80:483-485; 1984. 4. Bolme, P.; Fuxe, K. Pharmacological studies on the hypotensive effects of clonidine. Eur. J. Pharmacol. 13:168-174; 1971. 5. Fredholm, B.; Jansen, I.; Edvinsson, L. Neuropeptide Y is a potent inhibitor of cyclic AMP accumulation in feline cerebral blood vessels. Acta Physiol. Scand. 124:467-469; 1985. 6. Fuxe, K.; Agnati, L.; Harfstrand, A.; Zini, I.; Tatemotor, K.; Pich, E.; Hokfelt, T.; Mutt, V.; Terenius, L. Central administration of neuropeptide Y induces hypotension bradypnea and EEG synchronization in the rat. Acta Physiol. Scand. 118:189-192; 1983. 7. Gellai, M.; Ruffolo, R. R., Jr. Renal effects of selective alpha-1 and alpha-2 adrenoceptor agonists in conscious, normotensive rats. J. Pharmacol. Exp. Ther. 240:723-728; 1987. 8. Hackenthal, E.; Aktories, K.; Jakobs, K. H.; Lang, R. E. Neuropeptide Y inhibits renin release by a pertussis toxin-sensitive mechanism. Am. J. Physiol. 252:F543-F550; 1987. 9. Hakanson, R.; Wahlestedt, C.; Ekblad, E.; Edvinsson, L.; Sundler, F. Neuropeptide Y: Coexistence with noradrenaline: Functional implications. Prog. Brain Res. 68:279-287; 1986. 10. Harfstrand, A. Intraventricular administration of neuropeptide Y (NPY) induces hypotension, bradycardia and bradypnoea in the awake unrestrained male rat. Counteraction by NPY-induced feeding behav-

iours. Acta Physiol. Scand. 128:121-123; 1986. 11. Kassis, S.; Olasmaa, M.; Terenius, L.; Fishman, P. Neuropeptide Y inhibits cardiac adenylate cyclase through a pertussis toxin-sensitive G protein. J. Biol. Chem. 262:3429-3431; 1987. 12. Koepke, J.; DiBona, G. Central adrenergic receptor control of renal function in conscious hypertensive rats. Hypertension 8:133-141; 1986. 13. Lundberg, J. M.; Tatemotor, K. Pancreatic polypeptide family (APP, BPP, NPY and PYY) in relation to sympathetic vasoconstriction resistant to alpha-adrenoceptor blockade. Acta Physiol. Scand. 116: 393--402; 1982. 14. Lundberg, J.; Terenius, L.; Hokfelt, T.; Martling, C.; Tatemotor, K.; Mutt, V.; Polak, J.; Bloom, S.; Goldstein, M. Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effect of NPY on sympathetic function. Acta Physiol. Scand. 116: 477-480; 1982. 15. Pedraza-Chaverri, J.; Altorre-Gonzalez, M. C.; Ibarra-Rubio, M. E.; Pena, J. C.; Garcia-Sainz, J. A. Effect of pertussis toxin on the adrenergic regulation of plasma renin activity. Life Sci. 35:16381643; 1984. 16. Smyth, D. D.; Umemura, S.; Pettinger, W. A. Alpha2-adrenoceptor antagonism of vasopressin induced changes in sodium excretion. Am. J. Physiol. 248:F767-F772; 1985. 17. Strandhoy, J. W.; Morris, M.; Buckalew, V. M., Jr. Renal effects of the antihypertensive, guanabenz, in the dog. J. Pharmacol. Exp. Ther. 221:347-352; 1982. 18. Tatemoto, K.; Carlquist, M.; Mutt, V. Neuropeptide Y--a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 296:659-660; 1982. 19. Wahlestedt, C.; Edvinsson, L.; Ekblad, E.; Hakanson, R. Neuropeptide Y potentiates noradrenaline-evoked vasoconstriction: Mode of action. J. Pharmacol. Exp. Ther. 234:735-741; 1985.