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Sensors for antidiuresis and thirst—osmoreceptors or CSF sodium detectors?
Brain Research, 141 (1978) 89-103 © Elsevier/North-Holland Biomedical Press
89
SENSORS F O R ANTIDIURESIS A N D T H 1 R S T - - O S M O R E C E P T O R S OR CSF SODIUM D E T E C T O R S ?
M. J. McKINLEY, D. A. DENTON and R. S. WEISINGER
Howard Florey Institute of Experimental Physiology and Medicine, University t~f Melbourne, Parkville, Victoria 3052 (Australia) (Accepted May 18th, 1977)
SUMMARY
Change in sodium concentration of lateral ventricle CSF caused by intracarotid infusion of hypertonic solutions was measured in conscious sheep. Intracarotid infusion (1.6 ml/min) of 1 M NaC1, 2 M sucrose or 4.6 M or 2 M urea caused progressive increase of CSF sodium concentration, whereas 2 M glucose, 2 M galactose or 0.15 M NaCI did not. Of these solutions, only intracarotid 1 M NaCI or 2 M sucrose caused rapid water intake or rapid decrease in free water clearance. 2 M urea caused relatively slow antidiuresis and no water intake. 4.6 M urea which produced the largest rise of CSF [Na] caused slow antidiuresis and inconsistent small water intake. Infusion into a lateral ventricle (0.05 ml/min) of 0.35 M NaC1 or 0.7 M sucrose, or fructose, made up in artificial CSF (0.15 M Na) or 0.5 M NaC1 alone, all rapidly elicited an antidiuresis and water drinking, whereas intraventricular infusion of pure non-saline 1 M sucrose or 0.7 M urea in CSF was ineffective. Intraventricular 0.35 M NaC1 in CSF caused greater antidiuretic and dipsogenic effects than intraventricular 0.7 M sucrose or fructose in CSF. It is postulated that a dual osmoreceptor-sodium sensor system may participate in regulating antidiuretic hormone secretion and thirst, and that the osmoreceptor system mediates the rapid antidiuresis and water drinking caused by intracarotid 1 M NaCI or 2 M sucrose, and is probably located in a brain region without a blood-brain barrier.
INTRODUCTION
Uncertainty exists at present as to whether brain osmoreceptors40, 44 or periventricular sodium sensors3, 32 are responsible for initiating antidiuretic hormone (ADH) secretion and thirst21, 37. The osmoreceptor concept is derived from evidence that increase in the blood osmotic pressure caused by systemic administration of hypertonic solutions of solutes which penetrate relatively slowly into cells (e.g. NaCI
90 or sucrose) causes antidiuresis and thirst in dogs, whereas administration of hypertonic solutions of more diffusible solutes (e.g. urea or dextrose) is far less effective19,ee, 40. The mode of action of osmoreceptors has therefore been assumed to depend on shrinkage and expansion resulting from osmotically obligatory water movement in and out of these cells. The site of the osmoreceptor cells which control A D H secretion is not precisely defined though a substantial body of experimental evidence derived from the dog and cat would most favour functional localization in the supraoptic region2a,38, 45. However, data in other species suggests that the osmoreceptors are separated from the neurosecretory cells of the supraoptic nucleus, possibly in its perinuclear zone 2°,41 or elsewhere in the brain 34. A locational distinction between thirst and ADH-mediating osmoreceptors has also been made ~7,34. Andersson and colleagues, however, have made a number of observations in conscious goats which they consider incompatible with a simple osmoreceptor viewpoint (i) Whereas intracarotid infusions of hypertonic solutions of NaCl, sucrose and fructose cause antidiuresis and thirst 17,31, only hypertonic NaC1 elicits these effects when infused into the third ventricle. The hypertonic saccharide solutions are ineffective1,5,17. (ii) Reduction of cerebrospinal fluid (CSF) sodium concentration, but not tonicity, by intraventricular infusion of slightly hypertonic sucrose, fructose or glucose solution represses the antidiuretic, dipsogenic and natriuretic effects of intracarotid hypertonic NaC1 infusionsa,3L (iii) Rapid intracarotid infusions of slightly hypertonic glucose or galactose solutions induce a water diuresis which is abolished by intraventricular hypertonic NaCl a~. (iv) Since an effective blood-brain barrier exists to urea, it could be expected that intracarotid hypertonic urea infusions would dehydrate osmoreceptors if they were situated within the blood-brain barrier and cause thirst and antidiuresis z, 17 Such effects are not observed. Consequently, a periventricular sensor responding to change in CSF sodium concentration, in the vicinity of the third ventricle, has been proposed to mediate A D H secretion, water intake and Na excretion~,4,aa. The aim of the present experiments was to examine the feasibility of periventricular Na receptors being involved in water balance regulation in the sheep. If such receptor elements exist, appropriate changes in Na concentration of CSF should accompany A D H secretion and thirst produced by systemic hypertonicity. Therefore, change in ventricular CSF Na concentration has been measured during intracarotid infusion of various hypertonic solutions and related to the antidiuretic and dipsogenic effects of such infusions. Effects of infusion of various hypertonic artificial CSF solutions into a lateral ventricle on water intake and excretion by the kidney have also been studied. METHODS Experiments were carried out on a total of 18 Merino-cross ewes of body weight
91 32-38 kg. These animals were housed in metabolism cages and were fed a diet of mixed oaten-lucerne chaff (1 kg/day) at 1630 h and water was provided ad libitum.
Surgical procedures Prior to experimentation, each animal was surgically prepared with the carotid arteries exteriorised in bilateral cervical skin loops 16 and 17 g stainless steel guide tubes were permanently implanted over one or both lateral ventricles at the level of the interventricular foramenza. The position of the guide tubes was verified by X-ray after injection of contrast medium (sodium iodophendylate; Myodil, Glaxo) into a lateral ventricle. All surgical procedures were carried out using general anaesthesia (sodium thiopentone and maintained with fluothane/oxygen mixture) and aseptic techniques. Animals recovered well from these procedures and had resumed normal food and water intakes in 2-4 days. A recovery period of 2-3 weeks was allowed before commencing experiments. During this time the sheep became accustomed to laboratory personnel and procedures.
CSF sampling and intraventricular infusion Collection of CSF from a lateral ventricle was made by inserting an inner needle of appropriate length into the guide tube and puncturing the lateral ventricle. CSF (1 ml per sample) was siphoned by gravity through polyethylene tubing fitted to the needle. Infusion into a lateral ventricle was made by inserting an inner needle of appropriate length into the guide tube and puncturing the lateral ventricle as evidenced by the withdrawal of a few drops of CSF. Infusion of artificial CSF at 0.05 ml/min (Sage Instruments syringe pump, Model 351) was then made into the lateral ventricle via polyethylene tubing fitted to the inner needle. Solutions infused into the lateral ventricle were made up in an artificial CSF. Pure solutions of 1 M sucrose and 0.5 M NaCI were also tested. The composition of these solutions is shown in Table I. Solutions were made with pyrogen-free distilled water and sterilized by millipore filtration or autoclave. Sodium and potassium concentration and osmolality were routinely checked.
Intracarotid infusions Intracarotid infusions were made through a polyethylene cannula inserted into the carotid artery prior to commencement of an experiment. At the same time a polyethylene cannula was inserted into the jugular vein ipsilateral to the carotid cannula in order to sample jugular blood (6 ml/sample). Cannulae were filled with heparinised isotonic saline when not in use. During intracarotid infusions the carotid artery contralateral to the infusion was occluded by a pneumatic cuff to allow essentially bilateral distribution of the infusate in the brain6, 7. Where CSF was sampled during intracarotid infusion it was always withdrawn from the lateral ventricle on the side of the infusion. Solutions of sterile 4.6 M urea, 2 M sucrose, glucose, galactose, urea, 1 M NaCI or 0.15 M NaC1 were infused at a rate of 1.6 ml/min into the carotid artery using a motor driven syringe pump (Palmer, England).
92
TABLE 1
Molar concentrations of solutions b!fused into the lateral ventricle (raM) Solution
Na
K
Ca
Mg
CI
SucroseFructose Urea
Isotonic artificial CSF 0.35 M NaC1 CSF 0.7 M Sucrose CSF 0.7 M Fructose CSF 0.7 M Urea CSF 1 M Sucrose Natural sheep CSF*
150 500 150 150 150 0 150
2.8 2.8 2.8 2.8 2.8 0 2.8
1.2 1.2 1.2 1.2 1.2 0 1.2
0.9 0.9 0.9 0.9 0.9 0 0.9
157 507 157 157 t57 0 --
0 0 700 0 0 1000 --
0 0 0 700 0 0 --
0 0 0 0 700 0
* From Mouw et al. '~7. Note NaHCO3 was not included in artificial CSF.
Chemical analyses S o d i u m a n d p o t a s s i u m concentrations of C S F , p l a s m a a n d urine were m e a s u r e d with a Technicon A u t o a n a l y s e r . O s m o l a l i t y was measured with a K n a u e r H a l b m i k r o osmometer.
Experimental protocols [i] Effect o f intracarotid infusions of hypertonic solutions on C S F Na concentration, water intake and urineflow. A sample o f C S F a n d p l a s m a was o b t a i n e d a n d then within two minutes the infusion o f test solution into the c a r o t i d a r t e r y was c o m menced. Twenty-five minutes later the C S F a n d p l a s m a were again sampled. Then the infusion was stopped. In the case o f 4.6 M urea, 2 M urea, 2 M sucrose or 1 M NaCI further experiments were carried out to examine the time course o f effect. Thus i n t r a c a r o t i d infusions were m a d e for 30 min with C S F a n d p l a s m a s a m p l e d at 0, 10, 20 a n d 30 min after c o m m e n c e m e n t o f infusion. In two separate sets o f experiments n o t involving C S F sampling, the effect o f these infusions on water i n t a k e a n d urine flow was tested. (a) In water-replete animals, allowed continual access to water, the volume o f water d r u n k during 25 min o f i n t r a c a r o t i d infusion was measured. Experiments were carried o u t 18-22 h after the feeding o f the previous day. (b) The effect on urine flow was m e a s u r e d in animals u n d e r g o i n g a water diuresis i n d u c e d by i n t r a r u m i n a l a d m i n i s t r a t i o n o f two litres o f tepid water 80-100 min p r i o r to m a k i n g the infusion. Urine collections o f 10 min d u r a t i o n were started 20 min before c o m m e n c i n g the i n t r a c a r o t i d infusion a n d c o n t i n u e d for 90 min. D u r a t i o n o f infusion was 25 min. Urine was collected via a b l a d d e r catheter, a n d b l o o d samples (6 ml) were o b t a i n e d f r o m the contralaterai c a r o t i d artery. Renal free water clearance was calculated for the 10 min p r i o r to infusion, 10 a n d 20 min after c o m m e n c i n g the infusion, a n d 15 a n d 35 min after s t o p p i n g infusions.
[ii] Effect of infusion of hypertonic solutions into the lateral ventricle on water intake and urine flow. In water-replete animals, allowed free access to water, the h y p e r t o n i c solution u n d e r test was infused at 0.05 m l / m i n into a lateral ventricle for 20
93
O
¢Q
M~NdN~
~Z~ b,
~
ovV
94 min and the water intake measured. The experiments were carried out 18-22 h after the animals had been fed. In order to study urine flow, further experiments were carried out on animals undergoing a water diuresis induced by a prior intraruminal load of two litres of tepid water. The hypertonic solution under test was infused into a lateral ventricle for 10 min at 0.05 ml/min. Urine was collected at 10-min intervals from a bladder catheter and blood samples were obtained from a carotid artery. Renal free water clearance was determined 10 min prior to infusion and 20-30, 30-40 and 50-60 rain after commencing the intraventricular infusion. In 6 experiments, arterial blood pressure was recorded from a cannula inserted into the carotid artery and coupled to a Statham P23Db transducer and Offner type RS dynograph.
Statistical analysis Results are expressed throughout as mean ~: S.E.M. In order to test the effect of a particular infused solution, pre-infusion values have been compared to values obtained during or after infusions by the paired t-test. Changes caused by the various solutions infused have been compared by Student t-test. RESULTS
Effect of carotid artery infusion of hypertonic solutions on CSF Na concentration Infusion into the carotid artery of 2 M sucrose, 2 M urea or 1 M NaCI at !.6 ml/min for 25 min caused significant increases in CSF Na concentration, while infusion of 2 M glucose, 2 M galactose and 0.15 M NaC1 caused no change (Table II). The increase in CSF Na concentration caused by 2 M sucrose infusion was significantly greater than that observed with 2 M urea (P < 0.05) or 1 M NaCI (P < 0.05). On investigation of the time course of increase in CSF Na concentration caused by intracarotid infusions (Fig. 1), it was found that 4.6 M urea caused a larger and more rapid increase in CSF Na concentration than did 2 M sucrose infusion. However, 2 M sucrose caused more rapid increase in CSF Na than did either 2 M urea or 1 M NaCl. These latter two infusions caused similar increases in CSF Na concentration during intracarotid infusion for 30 min. Plasma Na concentration in jugular blood increased with the 1 M NaC1 infusion, was not significantly changed by 4.6 M or 2 M urea or 0.15 M NaCt, while 2 M sucrose, 2 M glucose or 2 M galactose significantly reduced plasma Na concentration (Table II and Fig. 1). Changes in CSF and plasma K concentration are also shown in Table II.
Effect of carotid artery infusion of hypertonic solutions on water intake Infusion into the carotid artery at 1.6 ml/min for 25 min of 1 M NaCI or 2 M sucrose caused water intake to commence within 2-4 rain of starting the infusion, and it continued sporadically during the duration of infusion in all experimental trials, while 4.6 M urea infusion caused water drinking in only three of 10 trials (Table Ill). Intracarotid infusions of 2 M glucose, 2 M urea or 0.15 M NaC1 were completely ineffective in causing water drinking, although 1 M NaCI infused at 1.6 ml/min still elicited water drinking when it was infused into the carotid artery in combination with 2 M urea infusion at 1.6 ml/min.
95 • 4.6M Urea
MeaniSEM
• 2 M Sucrose ~' I M NaCI O 2 M Urea oo.lsM N a C l
Change
n=4
I ~1_ ~
~
I
.
~
in CSF
[S~ mM
5 3
~
~
-4_s I0 Duration
20 of
Infusion
30 (min)
Fig. 1. Effect of intracarotid infusion of 0.15 M NaC1, 1 M NaC1 2 M sucrose, 2 M urea or 4.6 M urea at 1.6 ml/min for 30 rain on CSF and jugular vein plasma Na concentration. A significant change from final pre-infusion value is denoted by * P < 0.05, t P < 0.01, (paired t-test). Other P levels were obtained by Student t-test. TABLE III
Volume o f water drunk during intracarotid infusion of various hypertonic solutions at 1.6 ml/min for 25 min Solution infused
Number of trials
Trials exhibiting drinking responses
Water intake (ml, mean q- S.E.M.)
1 M NaC1 0.15 M NaC1 2 M Sucrose 4.6 M Urea 2 M Urea 2 M Glucose 1 M NaCI + 2 M Urea
8 7 8 10 4 4 4
8 0 8 3 0 0 4
469 0 531 185 0 0 569
* Significantly different from infusion of 0.15 M NaCI (P < 0.01).
:~ 86* -4- 108' :~ 103
-4- 146"
96 • 4.6 M Urea [5] • 2 M Sucrose['4]
Intracarotid infusion
[41 RENAL CH2O ml/min
0
t0
20
30
Time
40
50
60
70
(rain)
Fig. 2. Effect of intracarotid infusions of hypertonic solutions on renal free water clearance (CH~0) of water-loaded sheep. Infusion rate was 1.6 ml/min for 25 min. A significant reduction in the mean free water clearance from final pre-infusion value is denoted b), * P -< 0.05, t P -< 0.01 (paired t-test). Number of trials are indicated in parentheses. Other P levels were obtained by Student t-test. TABLE IV
Volume of water drunk during infusion of various hypertonic solutions into the lateral ventricle at 0.05 ml/min for 20 rain Solution infused
Number of trials
Trials with drinking responses
Water intake (ml, mean ~- S.E.M.)
0.7 M Fructose CSF 0.7 M Sucrose CSF 0.7 M Urea CSF 0.35 M N a C I CSF 0.5 M N a C I CSF (control) I MSucrose
4 8 7 12 8 7 7
4 8 2 12 8 1 3
330 305 64 946 741 17 173
± ! ± :k ± q. ~
117",** 72*,** 53** 105" 111" 15"* 100'*
* Significantly different from infusion of CSF (control) (P < 0.01 Student t-test). ** Significantly less than with infusion of 0.35 M NaCI CSF (P < 0.01 Student t-test).
97 x .5M NaCI [4] *-TM Urea CSF [4] IB,'/M
~rlll~tnQa
I'bC~: [A1
RENAL CH2C mi/mii
t0
20
30
40 50 Time (rnin)
60
70
Fig. 3. Effect of infusions of hypertonic solutions into the lateral cerebral ventricle on renal free water clearance (CH~o) of water-loaded sheep. Infusion rate was 0.05 ml/min for 10 rain. A significant reduction in the mean free water clearance from the final pre-infusion value is denoted by * P < 0.05, t P < 0.01 (paired t-test). Number of trials are indicated in parentheses. Other P levels were obtained by Stadent t-test.
Effect of intracarotid infusion of hypertonic solutions on renal free water clearance in water-loaded sheep Intraearotid infusion of 1 M N a C I or 2 M sucrose at 1.6 ml/min for 25 rain caused a significant reduction in renal free water clearance below control level 10-20 rain after c o m m e n c e m e n t o f infusion. Intracarotid infusion of 4.6 M or 2 M urea also caused reduction in free water clearance but this response was slower than with 1 M N a C I or 2 M sucrose in that the effect was n o t significant until 20-30 min after c o m m e n c e m e n t o f infusion. The reduction in free water clearance caused by intracarotid 2 M sucrose was also significantly greater (P < 0.05) than with 4.6 M or 2 M urea 20-30 min after c o m m e n c e m e n t of infusion (Fig. 2). Intracarotid 2 M glucose or 0.15 M NaC1 infusion caused no significant antidiuretic effects.
Effect of infusion of hypertonic solutions into the lateral ventricle on water intake in normal sheep and renal free water clearance in water-loaded sheep Solutions o f 0.35 M NaC1 in artificial C S F (total N a concentration 0.5 M ) or 0.7 M sucrose or fructose, in artificial C S F (Na concentration 0.15 M ) and pure 0.5 M
98 TABLE V Summary of changes in / NaJ of CSF, water intake and reduction in free water clearance caused by intracarotid or intraventricular infusion of various hypertonic solutions Solution htfused intraearotid
Measured d CSF f Naj m M
Water intake ml (frequency)
Reductionin CH2O
0.15 M NaCI
1 M NaC1 2 M Sucrose 2 M Urea 2 M Glucose 4.6 M Urea
0
0
0
+2.5 +4 + 2.5 0 +7
470(8/8) 531(8/8) 0 0 185(3/10)
++ -t -I + 0 -t--
Solution infused intraventricular
Expected A CSF [ Na]
Water intake ml (frequency)
Reduction in C~2o
0.7 M Fructose CSF 0.7 M Sucrose CSF 0.7 M Urea CSF 0.35 M NaCl CSF 0.5 M NaC1 CSF 1 M Sucrose
0 0 0 f ~ 0 ~
330(4/4) 305(8/8) 64(2/7) 946(12/12) 741(8/8) 17(1/7) 173(3/7)
+ 0 + -t ~- + 0 0
NaC1 caused water drinking within 2-6 min of commencement of infusion at 0.05 ml/min for 20 rain into the lateral ventricle (Table IV). Intraventricular infusion of 0.7 M urea in artificial CSF was inconsistent, inducing water intake in only 2 of 7 trials, while intraventricular infusion of pure non-saline 1 M sucrose was effective in causing water intake in only 3 of 7 trials. Isotonic artificial CSF caused drinking in 1 of 7 trials. The volume of water drunk with 0.35 M NaC1 in artificial CSF or 0.5 M NaC1 was significantly greater than with the infusions of 0.7 M sucrose, fructose, or urea in artificial CSF (P < 0.01). Intraventricular infusions of 0.35 M NaC1 CSF (total [Na] of 0.5 M), 0.7 M sucrose or fructose in artificial CSF or 0.5 M NaC1 caused marked reduction in free water clearance although infusions of 0.35 M NaCl in CSF or0.5 M NaC1 were more effective in reducing free water clearance when compared to 0.7 M sucrose or fructose CSF. Isotonic artificial CSF (0.15 M Na), 0.7 M urea CSF or pure 1 M sucrose were not effective antidiuretic agents when infused into a lateral ventricle for l0 min. Results are shown in Fig. 3. In experiments in which arterial blood pressure was continuously recorded, intraventricular infusion of 0.5 M NaC1 CSF (n = 3) caused a gradual increase of blood pressure of 5-15 m m H g which persisted for 15-30 min after stopping infusions. Intraventricular infusion of 1 M sucrose CSF did not alter arterial blood pressure (n = 3). DISCUSSION
It is generally accepted that thirst and antidiuretic hormone secretion are regulated in the main by the effective osmotic pressure of the body fluids. However, as stated in the introduction, the location of receptor elements sensing change in effective
99 osmotic pressure of the body fluids is not clear and the actual nature of the receptor with regard to osmotic or sodium-specific stimulation is unresolved. Our results summarised in Table V show that CSF Na concentration does increase when osmolality of the blood supplying the brain is increased by intracarotid infusion of hypertonic solutions of NaC1 or sucrose which are both effective dipsogenic and antidiuretic stimuli. However, intracarotid infusion of 4.6 M or 2 M urea also significantly increased CSF Na concentration yet were much weaker antidiuretic and dipsogenic agents. Intracarotid 4.6 M urea caused the greatest increase in CSF Na concentration yet had much less effect than 2 M sucrose or 1 M NaC1 in eliciting thirst and antidiuresis. It could be expected that if periventricular sodium receptors were the sole mediation of the rapid water drinking and antidiuresis observed with hypertonic sucrose and NaC1, similar effects should have been observed with intracarotid hypertonic urea infusions, but such was not the case. Decerebration of cats with removal of choroid plexus and substantial periventricular tissue leaving only isolated hypothalamic islets does not abolish the antidiuretic response to hypertonicity 45, which also suggests that CSF Na concentration change is not the sole essential factor involved in mediating A D H secretion due to systemic hypertonicity. Injection into the forebrain of rats of hypertonic NaCI is a more effective stimulus to water drinking than intraventricular hypertonic NaC19, which also argues against a sensor for CSF [Na]. Intracarotid hypertonic glucose, galactose or isotonic NaC1 caused no significant change in CSF Na concentration nor any appreciable reduction in urine flow. This latter observation confirms that intracarotid infusion, or CSF sampling procedures per se did not change CSF Na concentration, water intake or urine flow. As CSF was withdrawn from the lateral ventricle, it could be argued that the sodium concentration of CSF in the third ventricle was not represented by that of the lateral ventricle CSF. This seems unlikely since the point of withdrawal was directly over the foramen of Monro, which freely conducts CSF from lateral to third ventricle. The natural direction of CSF flow is from lateral to third ventricle 15, and we have observed radiographically in sheep that 0.5 ml contrast medium injected into a lateral ventricle appears within 15 sec in the third ventricle. It is likely that the lateral ventricles and third ventricle have CSF of similar ionic composition due to the easy access of CSF from lateral to third ventricle. It was considered that intracarotid infusions of 2 M urea may have had some deleterious effect on sheep and the lack of drinking to this stimulus may have been due to such an effect. However, intracarotid infusion of 1 M NaC1 still elicited adequate water drinking when combined with intracarotid 2 M urea infusion, making it unlikely that a deleterious effect of 2 M urea infusion was the cause of the failure of this solution to induce water intake. The finding that intracarotid infusion of hypertonic sucrose, NaC1 or urea increased CSF sodium concentration, whereas hypertonic glucose did not, is in agreement with data from several species that a far more effective blood-CSF barrier exists for sucrose, NaCI and urea than for glucose or galactose11,lz,15. Since there is adequate evidence which shows that water passes rapidly across the choroid plexusS, 39
100 the increased CSF sodium concentration was probably caused by osmotic withdrawal of water from CSF to blood. Clinical and experimental observations show that intravascular addition of hypertonic solutions of sucrose, NaCI or urea reduces intracranial pressure 24,2~,42 because of osmotic withdrawal of brain water across a blood-brain barrier which exists for these molecules but not for glucose 1~,14,46. Some disagreement exists in the literature with regard to the permeability of the blood-brain barrier to urea. Using various methods, several authors 1~,29,46 have found the permeability of the blood-brain barrier to be approximately the same for urea, Na and sucrose while others found a reflection co-efficient for urea of 0.46 that of sucrose 18. Using concentrations of urea of 2 M and 4.6 M we have covered the range of equivalent amounts of urea needed for comparison with 2 M sucrose or 1 M NaCI with regard to osmotic effect at the blood-brain barrier. Thus, it is likely that intracarotid infusions of hypertonic urea as well as sucrose and NaCI increase the sodium concentration and therefore effective osmotic pressure of brain interstitial fluid in areas with a blood-brain barrier. However, only sucrose and NaC1 cause rapid antidiuresis and thirst. Therefore it is likely that the osmoreceptors which mediate these responses are situated in a region or regions of the brain lacking a blood-brain barrier, e.g. circumventricular structures such as the subfornical organ, supraoptic crest 43. The supraoptic nucleus appears to be within the blood-brain barrier 36. It is also relevant that combined ablation of the subfornical organ and supraoptic crest caused complete adipsia and disruption of A D H secretion in the goat, despite an intact supraoptico-neurohypophyseal systemL In the case of 2 M and 4.6 M urea in the present experiments, an antidiuretic effect began to be observed 20-30 min after commencement ofintracarotid infusion and 4.6 M u r e a caused water intake in 30 % of infusions. Other workers have reported delayed or weak dipsogenic and antidiuretic effects of intracarotid hypertonic urea infusion 17,47. It is unlikely that these effects were due to rapid osmotic water withdrawal from osmoreceptors prior to diffusion of urea into these cells because of the relatively slow responses. Rather, the explanation of this phenomenon may be that sensor elements exist which are situated in, or influenced by the osmotic pressure of brain regions which possess a blood-brain barrier to urea. Since infusion of hypertonic urea should increase both sodium concentration and osmolality of CSF and interstitial fluid in such regions, such sensors could be responding to either change of Na concentration or osmolality. Turning now to the data obtained by making slow infusions of hypertonic artificial CSF solutions into the lateral ventricle, and summarised in Table V, it was observed that antidiuresis and consistent water drinking was caused by intraventricular infusion of hypertonic NaC1, sucrose, fructose but not urea - - all made in artificial CSF. In the case of the saccharides it is unnecessary to invoke a CSF sodium sensor in explanation of the responses, as CSF Na concentration should not have increased. As the more diffusible urea did not cause these responses the effects are consistent with an osmoreceptor and analogous to results in other species ~°,34,~. The relative lack of effect of intraventricular infusion of pure non-saline l M sucrose may have been due to the fact that CSF sodium concentration had been reduced as we have previously suggested 26, and confirms in this sense the importance of the CSF sodium
101 chloride concentration in the functioning of osmoregulatory responses 32,3a. In addition to reduction of NaC1 concentration of CSF, infusion of pure sucrose solution not made up in artificial CSF into the ventricle will also reduce the concentration of other important ions (e.g. K, Ca, Mg). However, pure 0.5 M NaCI caused rapid antidiuresis and thirst without correction of K, Ca and Mg concentrations, suggesting that the Na concentration is most important for these responses. While it may seem that the lack of antidiuretic and dipsogenic responses to intraventricular infusion of pure non-saline hypertonic solutions observed by the Swedish workers1,4,17, may be explained by failure to maintain basal CSF NaC1 concentrations, we observed that intraventricular infusion of hypertonic NaCI caused significantly greater water intake and more prolonged antidiuresis than the equiosmolar saccharide artificial CSF solutions when infused into the lateral ventricle. Thus, a specific effect of NaCI independent of its osmotic properties appears to be exerted. Initially, it was considered that this differential effect which we had previously observed on drinking behaviourz6, might be due to the differences in accessibility of stimulants to the receptors 34, or be due to non-specific neuronal influences because of the high NaCI concentration used relative to physiological levels. The evidence favours the alternative explanation that, in addition to osmoreceptors, there are also sodium or chloride specific sensors involved in control of water balance. If differential accessibility of the hypertonic agent to the receptors were involved, it could be expected that the latency of the responses would be different and this seemed not to occur. Rather, there was greater duration of the antidiuretic and dipsogenic effects caused by intraventricular hypertonic sodium solution. Non-specific neuronal excitation also seems less likely since, if this were the case, other behavioural effects (e.g. panting, general disturbance, hunger) could have been expected similar to those observed when KC1 is administered into the ventricle ~0. In addition, dilution by CSF and interstitial fluid will considerably reduce the initial high concentrations of the infusates. Blood pressure was elevated slightly by intraventricular infusion of hypertonic NaC1. but was unaffected by intraventricular infusion of 0.7 M sucrose CSF, suggesting that the antidiuretic effects of the intraventricular infusions were not dependent on a pressor response. In summary, the changes in CSF sodium concentration suggest that initial effects on water intake and urine production caused by intracarotid infusions of various hypertonic solutions are more consistent with mediation by brain osmoreceptors located outside the blood-brain barrier system than by periventricular sodium receptors. However, the greater dipsogenic and antidiuretic effects of intraventricular hypertonic NaC1 CSF infusion compared to intraventricular infusion of other equiosmolar saccharide CSF solutions suggests the participation of sodium (or chloride) sensors within the blood-brain barrier. We speculate that a dual sensing system in the brain involving osmoreceptors and sodium sensors may be involved in regulating water balance in sheep.
102 ACKNOWLEDGEMENTS This work was supported by grants from the N a t i o n a l Health a n d Medical Research Council of Australia, the Laura Bushell Trust, a n d the A u s t r a l i a n K i d n e y Foundation.
REFERENCES 1 Andersson, B., Jobin, M. and Olsson, K., A study of thirst and other effects of an increased sodium concentration in the 3rd brain ventricle, Acta physiol, scand., 69 (1967) 29-36. 2 Andersson, B., Leksell, L. and Lishajko, F., Perturbations in fluid balance induced by medially placed forebrain lesions, Brain Research, 99 (1975) 261-275. 3 Andersson, B. and Olsson, K., Importance of sodium in central control of fluid homeostasis. In R.O. Scow (Ed.), Endocrinology. Proceedings of the 4th International Congress of Endocrinology, Excerpta Medica, Amsterdam, 1973, pp. 724-728. 4 Andersson, B. and Olsson, K., On central control of body fluid homeostasis, Condit. Reflex, 8 (1973) 147-160. 5 Andersson, B., Olsson, K. and Warner, R. G., Dissimilarities between the central control of thirst and the release of antidiuretic hormone, Acta physiol, stand., 71 (1967) 57-64. 6 Baldwin, B. A. and Bell, F. R., The anatomy of the central circulation of the sheep and ox. The dynamic distribution of the blood supplied by the carotid and vertebral arteries to cranial regions, J. Anat. (Lond.), 97 (1963) 203-215. 7 Beilharz, S., Bott, E., Denton, D. A. and Sabine, J.R., The effect of intracarotid infusions of 4 M-NaCI on the sodium drinking of sheep with a parotid fistula, J. PhysioL (Lond.), 178 (1965) 80-91. 8 Bering, E. A., Water exchange of central nervous system and cerebrospinal fluid, J. Neurosurg., 9 (1952) 275-287. 9 Blass, E. M., Evidence for basal forebrain osmoreceptors in rat, Brain Research, 82 (1974) 69-76. 10 Blass, E. M. and Epstein, A. N., A lateral preoptic osmosensitive zone for thirst in the rat, J. comp. physiol. Psychol., 76 (1971) 378-394. 11 Coppen, A. J., Abnormality of the blood cerebrospinal fluid barrier of patients suffering from depressive illness, J. NeuroL, 23 (1960) 156-161. 12 Coxon, R. V., Cerebrospinal fluid transport. In A. Lajtha and D. H. Ford (Eds.), Progress in Brain Research, Vol. 29, Brain Barrier Systems, Elsevier, Amsterdam, 1968, pp. 134-146. 13 Crone, C., Facilitated transfer of glucose from blood into brain tissue, J. Physiol. (Lond.), 181 (1965) 103-113. 14 Crone, C., The permeability of brain capillaries to non-electrolytes, Actaphysiol. scand., 67 (1965) 407-417. 15 Davson, H., Physiology of the Cerebrospinal Fluid, Churchill, London, 1967. 16 Denton, D. A., The study of sheep with permanent unilateral parotid fistulae, Quart. J. exp. PhysioL, 42 (1957) 72-95. 17 Eriksson, L., Fernandez, O. and Olsson, K., Differences in the antidiuretic response to intracarotid infusions of various hypertonic solutions in the conscious goat, Aeta physiol, scand., 83 (1971) 554-562. 18 Fenstermacher, J. D. and Johnson, J. A., Filtration and reflection coefficients of the rabbit bloodbrain barrier, Amer. J. PhysioL, 211 (1966) 341-346. 19 Gilman, A., The relation between blood osmotic pressure, fluid distribution and voluntary water intake, Amer. J. Physiol., 120 (1937) 323-328. 20 Hayward, J. N., Neural control of the posterior pituitary. In J. H. Comroe (Ed.), Annual Review of Physiology, VoL 37, Annual Reviews, Palo Alto, 1975, pp. 191-210. 21 Hayward, J. N. and Jennings, D. P., Osmosensitivity of hypothalamic neuroendocrine cells to intracarotid hypertonic D-glucose in the waking monkey, Brain Research, 57 (1973) 467-472. 22 Holmes, J. H. and Gregerson, M. I., Observations on drinking induced by hypertonic solutions, Amer. J. Physiol., 162 (1950) 326-337.
103 23 Jewell, P. A. and Verney, E. B., An experimental attempt to determine the site of the neurohypophyseal osmoreceptors in the dog, Phil. Trans. B, 240 (1957) 197-324. 24 Javid, M. and Anderson, J., The effect of urea on cerebrospinal fluid pressure in monkeys before and after bilateral nephrectomy, J. Lab. clin. Med., 53 (1959) 484-489. 25 Javid, M. and Settlage, P., Effect of urea on cerebrospinal fluid pressure in human subjects, J. Amer. reed. Ass., 160 (1956) 943-949. 26 McKinley, M. J., Blaine, E. H. and Denton, D. A., Brain osmoreceptors, cerebrospinal fluid electrolyte composition and thirst, Brain Research, 70 (1974) 532-537. 27 Mendelsohn, J., Feinberg, L. E. and Berman, J., Dissociation of diencephalic mechanisms controlling water intake and water retention in the rat, Amer. J. Physiol., 220 (1971) 1768-1774. 28 Mouw, D. R., Abraham, S. F., Blair-West, J. R., Coghlan, J. P., Denton, D. A., McKenzie, J. S., McKinley, M. J. and Scoggins, B. A., Brain receptors, renin secretion and renal sodium retention in conscious sheep, Amer. J. Physiol., 226 (1974) 56-62. 29 Oldendorf, W. H., Brain uptake of radiolabeiled amino-acids, amines, and hexoses after arterial injection, Amer. J. Physiol., 221 (1971) 1629-1639. 30 Olsson, K., Effects of slow infusions of KCI into the third brain ventricle, Acta physiol, scand., 77 (1969) 465-474. 31 Olsson, K., Dipsogenic effects of intracarotid infusions of various hyperosmolal solutions, Acta physiol, scand., 85 (1972) 517-522. 32 Olsson, K., Further evidence for the importance of CSF Na concentration in central control of fluid balance, Acta physiol, scand., 88 (1973) 183-188. 33 Olsson, K. and Kolmodin, R., Dependence of basic secretion of antidiuretic hormone on cerebrospinal fluid [Na], Acta physiol, scand., 91 (1974) 286--288. 34 Peck, J. W. and Blass, E. M., Localization of thirst and antidiuretic osmoreceptors by intracranial injections in rats, Amer. J. Physiol., 228 (1975) 1501-1509. 35 Peck, J. W. and Novin, D., Evidence that osmoreceptors mediating drinking in rabbits are in the lateral preoptic area, J. comp. physioL Psychol., 74 (1971) 134-147. 36 Rechardt, L., Electron microscopic and histochemical observations on the supraoptic nucleus of normal and dehydrated rats, Acta physiol, scand., SuppL, 329 (1969) 1-80. 37 Share, L. and Grosvenor, C. E., The neurohypophysis. In S. M. McCann (Ed.), MTP International Review of Science. Endocrine Physiology, Butterworths, London, 1975, pp. 1-29. 38 Suda, I., Koizumi, K. and Brooks, C. McC., Study of unitary activity in the supraoptic nucleus of the hypothalamus, Jap. J. PhysioL, 13 (1963) 374-385. 39 Sweet, W. H., Selverstone, B., Solloway, S. and Stetten, D., Studies of formation flow and absorption of cerebrospinal fluid. II. Studies with heavy water in the normal man, Surgical Forum, 1 (1950) 376-381. 40 Verney, E. B., The antidiuretic hormone and the factors which determine its release, Proc. roy. Soe. B., 135 (1947) 25-106. 41 Vincent, J. D., Arnauld, E. and Nicolescu-Catargi, A., Osmoreceptors and neurosecretory ceils in the supraoptic complex of the unanaesthetized monkey, Brain Research, 45 (1972) 278-281. 42 Weed, L. H. and McKibben, P. S., Pressure changes in the cerebrospinal fluid following intravenous injections of solutions of various concentrations, Amer. J. Physiol., 48 (1919) 512-530. 43 Weindl, A. and Joynt, R. J., The median eminence as a circumventricular organ. In K. M. Knigge, D. E. Scott and A. Weindl (Eds.), Brain-Endocrine Interaction, Karger, Basel, 1972, pp. 280-297. 44 Wolf, A. V., Osmometric analysis of thirst in man and dog, Amer. J. Physiol., 161 (1950) 75-86. 45 Woods, J. W., Bard, P. and Bleier, R., Functional capacity of the deafferented hypothalamus: water balance and responses to osmotic stimuli in the decerebrate cat and rat, J. NeurophysioL, 29 (1966) 751-767. 46 Yudilevich, D. L. and de Rose, N., Blood-brain transfer of glucose and other molecules measured by rapid indicator dilution, Amer. J. PhysioL, 220 (1971) 841-846. 47 Zuidema, G. D. and Clarke, N. P., Central localization of the osmotic control centre, Amer. J. PhysioL, 188 (1957) 616-618.
89
SENSORS F O R ANTIDIURESIS A N D T H 1 R S T - - O S M O R E C E P T O R S OR CSF SODIUM D E T E C T O R S ?
M. J. McKINLEY, D. A. DENTON and R. S. WEISINGER
Howard Florey Institute of Experimental Physiology and Medicine, University t~f Melbourne, Parkville, Victoria 3052 (Australia) (Accepted May 18th, 1977)
SUMMARY
Change in sodium concentration of lateral ventricle CSF caused by intracarotid infusion of hypertonic solutions was measured in conscious sheep. Intracarotid infusion (1.6 ml/min) of 1 M NaC1, 2 M sucrose or 4.6 M or 2 M urea caused progressive increase of CSF sodium concentration, whereas 2 M glucose, 2 M galactose or 0.15 M NaCI did not. Of these solutions, only intracarotid 1 M NaCI or 2 M sucrose caused rapid water intake or rapid decrease in free water clearance. 2 M urea caused relatively slow antidiuresis and no water intake. 4.6 M urea which produced the largest rise of CSF [Na] caused slow antidiuresis and inconsistent small water intake. Infusion into a lateral ventricle (0.05 ml/min) of 0.35 M NaC1 or 0.7 M sucrose, or fructose, made up in artificial CSF (0.15 M Na) or 0.5 M NaC1 alone, all rapidly elicited an antidiuresis and water drinking, whereas intraventricular infusion of pure non-saline 1 M sucrose or 0.7 M urea in CSF was ineffective. Intraventricular 0.35 M NaC1 in CSF caused greater antidiuretic and dipsogenic effects than intraventricular 0.7 M sucrose or fructose in CSF. It is postulated that a dual osmoreceptor-sodium sensor system may participate in regulating antidiuretic hormone secretion and thirst, and that the osmoreceptor system mediates the rapid antidiuresis and water drinking caused by intracarotid 1 M NaCI or 2 M sucrose, and is probably located in a brain region without a blood-brain barrier.
INTRODUCTION
Uncertainty exists at present as to whether brain osmoreceptors40, 44 or periventricular sodium sensors3, 32 are responsible for initiating antidiuretic hormone (ADH) secretion and thirst21, 37. The osmoreceptor concept is derived from evidence that increase in the blood osmotic pressure caused by systemic administration of hypertonic solutions of solutes which penetrate relatively slowly into cells (e.g. NaCI
90 or sucrose) causes antidiuresis and thirst in dogs, whereas administration of hypertonic solutions of more diffusible solutes (e.g. urea or dextrose) is far less effective19,ee, 40. The mode of action of osmoreceptors has therefore been assumed to depend on shrinkage and expansion resulting from osmotically obligatory water movement in and out of these cells. The site of the osmoreceptor cells which control A D H secretion is not precisely defined though a substantial body of experimental evidence derived from the dog and cat would most favour functional localization in the supraoptic region2a,38, 45. However, data in other species suggests that the osmoreceptors are separated from the neurosecretory cells of the supraoptic nucleus, possibly in its perinuclear zone 2°,41 or elsewhere in the brain 34. A locational distinction between thirst and ADH-mediating osmoreceptors has also been made ~7,34. Andersson and colleagues, however, have made a number of observations in conscious goats which they consider incompatible with a simple osmoreceptor viewpoint (i) Whereas intracarotid infusions of hypertonic solutions of NaCl, sucrose and fructose cause antidiuresis and thirst 17,31, only hypertonic NaC1 elicits these effects when infused into the third ventricle. The hypertonic saccharide solutions are ineffective1,5,17. (ii) Reduction of cerebrospinal fluid (CSF) sodium concentration, but not tonicity, by intraventricular infusion of slightly hypertonic sucrose, fructose or glucose solution represses the antidiuretic, dipsogenic and natriuretic effects of intracarotid hypertonic NaC1 infusionsa,3L (iii) Rapid intracarotid infusions of slightly hypertonic glucose or galactose solutions induce a water diuresis which is abolished by intraventricular hypertonic NaCl a~. (iv) Since an effective blood-brain barrier exists to urea, it could be expected that intracarotid hypertonic urea infusions would dehydrate osmoreceptors if they were situated within the blood-brain barrier and cause thirst and antidiuresis z, 17 Such effects are not observed. Consequently, a periventricular sensor responding to change in CSF sodium concentration, in the vicinity of the third ventricle, has been proposed to mediate A D H secretion, water intake and Na excretion~,4,aa. The aim of the present experiments was to examine the feasibility of periventricular Na receptors being involved in water balance regulation in the sheep. If such receptor elements exist, appropriate changes in Na concentration of CSF should accompany A D H secretion and thirst produced by systemic hypertonicity. Therefore, change in ventricular CSF Na concentration has been measured during intracarotid infusion of various hypertonic solutions and related to the antidiuretic and dipsogenic effects of such infusions. Effects of infusion of various hypertonic artificial CSF solutions into a lateral ventricle on water intake and excretion by the kidney have also been studied. METHODS Experiments were carried out on a total of 18 Merino-cross ewes of body weight
91 32-38 kg. These animals were housed in metabolism cages and were fed a diet of mixed oaten-lucerne chaff (1 kg/day) at 1630 h and water was provided ad libitum.
Surgical procedures Prior to experimentation, each animal was surgically prepared with the carotid arteries exteriorised in bilateral cervical skin loops 16 and 17 g stainless steel guide tubes were permanently implanted over one or both lateral ventricles at the level of the interventricular foramenza. The position of the guide tubes was verified by X-ray after injection of contrast medium (sodium iodophendylate; Myodil, Glaxo) into a lateral ventricle. All surgical procedures were carried out using general anaesthesia (sodium thiopentone and maintained with fluothane/oxygen mixture) and aseptic techniques. Animals recovered well from these procedures and had resumed normal food and water intakes in 2-4 days. A recovery period of 2-3 weeks was allowed before commencing experiments. During this time the sheep became accustomed to laboratory personnel and procedures.
CSF sampling and intraventricular infusion Collection of CSF from a lateral ventricle was made by inserting an inner needle of appropriate length into the guide tube and puncturing the lateral ventricle. CSF (1 ml per sample) was siphoned by gravity through polyethylene tubing fitted to the needle. Infusion into a lateral ventricle was made by inserting an inner needle of appropriate length into the guide tube and puncturing the lateral ventricle as evidenced by the withdrawal of a few drops of CSF. Infusion of artificial CSF at 0.05 ml/min (Sage Instruments syringe pump, Model 351) was then made into the lateral ventricle via polyethylene tubing fitted to the inner needle. Solutions infused into the lateral ventricle were made up in an artificial CSF. Pure solutions of 1 M sucrose and 0.5 M NaCI were also tested. The composition of these solutions is shown in Table I. Solutions were made with pyrogen-free distilled water and sterilized by millipore filtration or autoclave. Sodium and potassium concentration and osmolality were routinely checked.
Intracarotid infusions Intracarotid infusions were made through a polyethylene cannula inserted into the carotid artery prior to commencement of an experiment. At the same time a polyethylene cannula was inserted into the jugular vein ipsilateral to the carotid cannula in order to sample jugular blood (6 ml/sample). Cannulae were filled with heparinised isotonic saline when not in use. During intracarotid infusions the carotid artery contralateral to the infusion was occluded by a pneumatic cuff to allow essentially bilateral distribution of the infusate in the brain6, 7. Where CSF was sampled during intracarotid infusion it was always withdrawn from the lateral ventricle on the side of the infusion. Solutions of sterile 4.6 M urea, 2 M sucrose, glucose, galactose, urea, 1 M NaCI or 0.15 M NaC1 were infused at a rate of 1.6 ml/min into the carotid artery using a motor driven syringe pump (Palmer, England).
92
TABLE 1
Molar concentrations of solutions b!fused into the lateral ventricle (raM) Solution
Na
K
Ca
Mg
CI
SucroseFructose Urea
Isotonic artificial CSF 0.35 M NaC1 CSF 0.7 M Sucrose CSF 0.7 M Fructose CSF 0.7 M Urea CSF 1 M Sucrose Natural sheep CSF*
150 500 150 150 150 0 150
2.8 2.8 2.8 2.8 2.8 0 2.8
1.2 1.2 1.2 1.2 1.2 0 1.2
0.9 0.9 0.9 0.9 0.9 0 0.9
157 507 157 157 t57 0 --
0 0 700 0 0 1000 --
0 0 0 700 0 0 --
0 0 0 0 700 0
* From Mouw et al. '~7. Note NaHCO3 was not included in artificial CSF.
Chemical analyses S o d i u m a n d p o t a s s i u m concentrations of C S F , p l a s m a a n d urine were m e a s u r e d with a Technicon A u t o a n a l y s e r . O s m o l a l i t y was measured with a K n a u e r H a l b m i k r o osmometer.
Experimental protocols [i] Effect o f intracarotid infusions of hypertonic solutions on C S F Na concentration, water intake and urineflow. A sample o f C S F a n d p l a s m a was o b t a i n e d a n d then within two minutes the infusion o f test solution into the c a r o t i d a r t e r y was c o m menced. Twenty-five minutes later the C S F a n d p l a s m a were again sampled. Then the infusion was stopped. In the case o f 4.6 M urea, 2 M urea, 2 M sucrose or 1 M NaCI further experiments were carried out to examine the time course o f effect. Thus i n t r a c a r o t i d infusions were m a d e for 30 min with C S F a n d p l a s m a s a m p l e d at 0, 10, 20 a n d 30 min after c o m m e n c e m e n t o f infusion. In two separate sets o f experiments n o t involving C S F sampling, the effect o f these infusions on water i n t a k e a n d urine flow was tested. (a) In water-replete animals, allowed continual access to water, the volume o f water d r u n k during 25 min o f i n t r a c a r o t i d infusion was measured. Experiments were carried o u t 18-22 h after the feeding o f the previous day. (b) The effect on urine flow was m e a s u r e d in animals u n d e r g o i n g a water diuresis i n d u c e d by i n t r a r u m i n a l a d m i n i s t r a t i o n o f two litres o f tepid water 80-100 min p r i o r to m a k i n g the infusion. Urine collections o f 10 min d u r a t i o n were started 20 min before c o m m e n c i n g the i n t r a c a r o t i d infusion a n d c o n t i n u e d for 90 min. D u r a t i o n o f infusion was 25 min. Urine was collected via a b l a d d e r catheter, a n d b l o o d samples (6 ml) were o b t a i n e d f r o m the contralaterai c a r o t i d artery. Renal free water clearance was calculated for the 10 min p r i o r to infusion, 10 a n d 20 min after c o m m e n c i n g the infusion, a n d 15 a n d 35 min after s t o p p i n g infusions.
[ii] Effect of infusion of hypertonic solutions into the lateral ventricle on water intake and urine flow. In water-replete animals, allowed free access to water, the h y p e r t o n i c solution u n d e r test was infused at 0.05 m l / m i n into a lateral ventricle for 20
93
O
¢Q
M~NdN~
~Z~ b,
~
ovV
94 min and the water intake measured. The experiments were carried out 18-22 h after the animals had been fed. In order to study urine flow, further experiments were carried out on animals undergoing a water diuresis induced by a prior intraruminal load of two litres of tepid water. The hypertonic solution under test was infused into a lateral ventricle for 10 min at 0.05 ml/min. Urine was collected at 10-min intervals from a bladder catheter and blood samples were obtained from a carotid artery. Renal free water clearance was determined 10 min prior to infusion and 20-30, 30-40 and 50-60 rain after commencing the intraventricular infusion. In 6 experiments, arterial blood pressure was recorded from a cannula inserted into the carotid artery and coupled to a Statham P23Db transducer and Offner type RS dynograph.
Statistical analysis Results are expressed throughout as mean ~: S.E.M. In order to test the effect of a particular infused solution, pre-infusion values have been compared to values obtained during or after infusions by the paired t-test. Changes caused by the various solutions infused have been compared by Student t-test. RESULTS
Effect of carotid artery infusion of hypertonic solutions on CSF Na concentration Infusion into the carotid artery of 2 M sucrose, 2 M urea or 1 M NaCI at !.6 ml/min for 25 min caused significant increases in CSF Na concentration, while infusion of 2 M glucose, 2 M galactose and 0.15 M NaC1 caused no change (Table II). The increase in CSF Na concentration caused by 2 M sucrose infusion was significantly greater than that observed with 2 M urea (P < 0.05) or 1 M NaCI (P < 0.05). On investigation of the time course of increase in CSF Na concentration caused by intracarotid infusions (Fig. 1), it was found that 4.6 M urea caused a larger and more rapid increase in CSF Na concentration than did 2 M sucrose infusion. However, 2 M sucrose caused more rapid increase in CSF Na than did either 2 M urea or 1 M NaCl. These latter two infusions caused similar increases in CSF Na concentration during intracarotid infusion for 30 min. Plasma Na concentration in jugular blood increased with the 1 M NaC1 infusion, was not significantly changed by 4.6 M or 2 M urea or 0.15 M NaCt, while 2 M sucrose, 2 M glucose or 2 M galactose significantly reduced plasma Na concentration (Table II and Fig. 1). Changes in CSF and plasma K concentration are also shown in Table II.
Effect of carotid artery infusion of hypertonic solutions on water intake Infusion into the carotid artery at 1.6 ml/min for 25 min of 1 M NaCI or 2 M sucrose caused water intake to commence within 2-4 rain of starting the infusion, and it continued sporadically during the duration of infusion in all experimental trials, while 4.6 M urea infusion caused water drinking in only three of 10 trials (Table Ill). Intracarotid infusions of 2 M glucose, 2 M urea or 0.15 M NaC1 were completely ineffective in causing water drinking, although 1 M NaCI infused at 1.6 ml/min still elicited water drinking when it was infused into the carotid artery in combination with 2 M urea infusion at 1.6 ml/min.
95 • 4.6M Urea
MeaniSEM
• 2 M Sucrose ~' I M NaCI O 2 M Urea oo.lsM N a C l
Change
n=4
I ~1_ ~
~
I
.
~
in CSF
[S~ mM
5 3
~
~
-4_s I0 Duration
20 of
Infusion
30 (min)
Fig. 1. Effect of intracarotid infusion of 0.15 M NaC1, 1 M NaC1 2 M sucrose, 2 M urea or 4.6 M urea at 1.6 ml/min for 30 rain on CSF and jugular vein plasma Na concentration. A significant change from final pre-infusion value is denoted by * P < 0.05, t P < 0.01, (paired t-test). Other P levels were obtained by Student t-test. TABLE III
Volume o f water drunk during intracarotid infusion of various hypertonic solutions at 1.6 ml/min for 25 min Solution infused
Number of trials
Trials exhibiting drinking responses
Water intake (ml, mean q- S.E.M.)
1 M NaC1 0.15 M NaC1 2 M Sucrose 4.6 M Urea 2 M Urea 2 M Glucose 1 M NaCI + 2 M Urea
8 7 8 10 4 4 4
8 0 8 3 0 0 4
469 0 531 185 0 0 569
* Significantly different from infusion of 0.15 M NaCI (P < 0.01).
:~ 86* -4- 108' :~ 103
-4- 146"
96 • 4.6 M Urea [5] • 2 M Sucrose['4]
Intracarotid infusion
[41 RENAL CH2O ml/min
0
t0
20
30
Time
40
50
60
70
(rain)
Fig. 2. Effect of intracarotid infusions of hypertonic solutions on renal free water clearance (CH~0) of water-loaded sheep. Infusion rate was 1.6 ml/min for 25 min. A significant reduction in the mean free water clearance from final pre-infusion value is denoted b), * P -< 0.05, t P -< 0.01 (paired t-test). Number of trials are indicated in parentheses. Other P levels were obtained by Student t-test. TABLE IV
Volume of water drunk during infusion of various hypertonic solutions into the lateral ventricle at 0.05 ml/min for 20 rain Solution infused
Number of trials
Trials with drinking responses
Water intake (ml, mean ~- S.E.M.)
0.7 M Fructose CSF 0.7 M Sucrose CSF 0.7 M Urea CSF 0.35 M N a C I CSF 0.5 M N a C I CSF (control) I MSucrose
4 8 7 12 8 7 7
4 8 2 12 8 1 3
330 305 64 946 741 17 173
± ! ± :k ± q. ~
117",** 72*,** 53** 105" 111" 15"* 100'*
* Significantly different from infusion of CSF (control) (P < 0.01 Student t-test). ** Significantly less than with infusion of 0.35 M NaCI CSF (P < 0.01 Student t-test).
97 x .5M NaCI [4] *-TM Urea CSF [4] IB,'/M
~rlll~tnQa
I'bC~: [A1
RENAL CH2C mi/mii
t0
20
30
40 50 Time (rnin)
60
70
Fig. 3. Effect of infusions of hypertonic solutions into the lateral cerebral ventricle on renal free water clearance (CH~o) of water-loaded sheep. Infusion rate was 0.05 ml/min for 10 rain. A significant reduction in the mean free water clearance from the final pre-infusion value is denoted by * P < 0.05, t P < 0.01 (paired t-test). Number of trials are indicated in parentheses. Other P levels were obtained by Stadent t-test.
Effect of intracarotid infusion of hypertonic solutions on renal free water clearance in water-loaded sheep Intraearotid infusion of 1 M N a C I or 2 M sucrose at 1.6 ml/min for 25 rain caused a significant reduction in renal free water clearance below control level 10-20 rain after c o m m e n c e m e n t o f infusion. Intracarotid infusion of 4.6 M or 2 M urea also caused reduction in free water clearance but this response was slower than with 1 M N a C I or 2 M sucrose in that the effect was n o t significant until 20-30 min after c o m m e n c e m e n t o f infusion. The reduction in free water clearance caused by intracarotid 2 M sucrose was also significantly greater (P < 0.05) than with 4.6 M or 2 M urea 20-30 min after c o m m e n c e m e n t of infusion (Fig. 2). Intracarotid 2 M glucose or 0.15 M NaC1 infusion caused no significant antidiuretic effects.
Effect of infusion of hypertonic solutions into the lateral ventricle on water intake in normal sheep and renal free water clearance in water-loaded sheep Solutions o f 0.35 M NaC1 in artificial C S F (total N a concentration 0.5 M ) or 0.7 M sucrose or fructose, in artificial C S F (Na concentration 0.15 M ) and pure 0.5 M
98 TABLE V Summary of changes in / NaJ of CSF, water intake and reduction in free water clearance caused by intracarotid or intraventricular infusion of various hypertonic solutions Solution htfused intraearotid
Measured d CSF f Naj m M
Water intake ml (frequency)
Reductionin CH2O
0.15 M NaCI
1 M NaC1 2 M Sucrose 2 M Urea 2 M Glucose 4.6 M Urea
0
0
0
+2.5 +4 + 2.5 0 +7
470(8/8) 531(8/8) 0 0 185(3/10)
++ -t -I + 0 -t--
Solution infused intraventricular
Expected A CSF [ Na]
Water intake ml (frequency)
Reduction in C~2o
0.7 M Fructose CSF 0.7 M Sucrose CSF 0.7 M Urea CSF 0.35 M NaCl CSF 0.5 M NaC1 CSF 1 M Sucrose
0 0 0 f ~ 0 ~
330(4/4) 305(8/8) 64(2/7) 946(12/12) 741(8/8) 17(1/7) 173(3/7)
+ 0 + -t ~- + 0 0
NaC1 caused water drinking within 2-6 min of commencement of infusion at 0.05 ml/min for 20 rain into the lateral ventricle (Table IV). Intraventricular infusion of 0.7 M urea in artificial CSF was inconsistent, inducing water intake in only 2 of 7 trials, while intraventricular infusion of pure non-saline 1 M sucrose was effective in causing water intake in only 3 of 7 trials. Isotonic artificial CSF caused drinking in 1 of 7 trials. The volume of water drunk with 0.35 M NaC1 in artificial CSF or 0.5 M NaC1 was significantly greater than with the infusions of 0.7 M sucrose, fructose, or urea in artificial CSF (P < 0.01). Intraventricular infusions of 0.35 M NaC1 CSF (total [Na] of 0.5 M), 0.7 M sucrose or fructose in artificial CSF or 0.5 M NaC1 caused marked reduction in free water clearance although infusions of 0.35 M NaCl in CSF or0.5 M NaC1 were more effective in reducing free water clearance when compared to 0.7 M sucrose or fructose CSF. Isotonic artificial CSF (0.15 M Na), 0.7 M urea CSF or pure 1 M sucrose were not effective antidiuretic agents when infused into a lateral ventricle for l0 min. Results are shown in Fig. 3. In experiments in which arterial blood pressure was continuously recorded, intraventricular infusion of 0.5 M NaC1 CSF (n = 3) caused a gradual increase of blood pressure of 5-15 m m H g which persisted for 15-30 min after stopping infusions. Intraventricular infusion of 1 M sucrose CSF did not alter arterial blood pressure (n = 3). DISCUSSION
It is generally accepted that thirst and antidiuretic hormone secretion are regulated in the main by the effective osmotic pressure of the body fluids. However, as stated in the introduction, the location of receptor elements sensing change in effective
99 osmotic pressure of the body fluids is not clear and the actual nature of the receptor with regard to osmotic or sodium-specific stimulation is unresolved. Our results summarised in Table V show that CSF Na concentration does increase when osmolality of the blood supplying the brain is increased by intracarotid infusion of hypertonic solutions of NaC1 or sucrose which are both effective dipsogenic and antidiuretic stimuli. However, intracarotid infusion of 4.6 M or 2 M urea also significantly increased CSF Na concentration yet were much weaker antidiuretic and dipsogenic agents. Intracarotid 4.6 M urea caused the greatest increase in CSF Na concentration yet had much less effect than 2 M sucrose or 1 M NaC1 in eliciting thirst and antidiuresis. It could be expected that if periventricular sodium receptors were the sole mediation of the rapid water drinking and antidiuresis observed with hypertonic sucrose and NaC1, similar effects should have been observed with intracarotid hypertonic urea infusions, but such was not the case. Decerebration of cats with removal of choroid plexus and substantial periventricular tissue leaving only isolated hypothalamic islets does not abolish the antidiuretic response to hypertonicity 45, which also suggests that CSF Na concentration change is not the sole essential factor involved in mediating A D H secretion due to systemic hypertonicity. Injection into the forebrain of rats of hypertonic NaCI is a more effective stimulus to water drinking than intraventricular hypertonic NaC19, which also argues against a sensor for CSF [Na]. Intracarotid hypertonic glucose, galactose or isotonic NaC1 caused no significant change in CSF Na concentration nor any appreciable reduction in urine flow. This latter observation confirms that intracarotid infusion, or CSF sampling procedures per se did not change CSF Na concentration, water intake or urine flow. As CSF was withdrawn from the lateral ventricle, it could be argued that the sodium concentration of CSF in the third ventricle was not represented by that of the lateral ventricle CSF. This seems unlikely since the point of withdrawal was directly over the foramen of Monro, which freely conducts CSF from lateral to third ventricle. The natural direction of CSF flow is from lateral to third ventricle 15, and we have observed radiographically in sheep that 0.5 ml contrast medium injected into a lateral ventricle appears within 15 sec in the third ventricle. It is likely that the lateral ventricles and third ventricle have CSF of similar ionic composition due to the easy access of CSF from lateral to third ventricle. It was considered that intracarotid infusions of 2 M urea may have had some deleterious effect on sheep and the lack of drinking to this stimulus may have been due to such an effect. However, intracarotid infusion of 1 M NaC1 still elicited adequate water drinking when combined with intracarotid 2 M urea infusion, making it unlikely that a deleterious effect of 2 M urea infusion was the cause of the failure of this solution to induce water intake. The finding that intracarotid infusion of hypertonic sucrose, NaC1 or urea increased CSF sodium concentration, whereas hypertonic glucose did not, is in agreement with data from several species that a far more effective blood-CSF barrier exists for sucrose, NaCI and urea than for glucose or galactose11,lz,15. Since there is adequate evidence which shows that water passes rapidly across the choroid plexusS, 39
100 the increased CSF sodium concentration was probably caused by osmotic withdrawal of water from CSF to blood. Clinical and experimental observations show that intravascular addition of hypertonic solutions of sucrose, NaCI or urea reduces intracranial pressure 24,2~,42 because of osmotic withdrawal of brain water across a blood-brain barrier which exists for these molecules but not for glucose 1~,14,46. Some disagreement exists in the literature with regard to the permeability of the blood-brain barrier to urea. Using various methods, several authors 1~,29,46 have found the permeability of the blood-brain barrier to be approximately the same for urea, Na and sucrose while others found a reflection co-efficient for urea of 0.46 that of sucrose 18. Using concentrations of urea of 2 M and 4.6 M we have covered the range of equivalent amounts of urea needed for comparison with 2 M sucrose or 1 M NaCI with regard to osmotic effect at the blood-brain barrier. Thus, it is likely that intracarotid infusions of hypertonic urea as well as sucrose and NaCI increase the sodium concentration and therefore effective osmotic pressure of brain interstitial fluid in areas with a blood-brain barrier. However, only sucrose and NaC1 cause rapid antidiuresis and thirst. Therefore it is likely that the osmoreceptors which mediate these responses are situated in a region or regions of the brain lacking a blood-brain barrier, e.g. circumventricular structures such as the subfornical organ, supraoptic crest 43. The supraoptic nucleus appears to be within the blood-brain barrier 36. It is also relevant that combined ablation of the subfornical organ and supraoptic crest caused complete adipsia and disruption of A D H secretion in the goat, despite an intact supraoptico-neurohypophyseal systemL In the case of 2 M and 4.6 M urea in the present experiments, an antidiuretic effect began to be observed 20-30 min after commencement ofintracarotid infusion and 4.6 M u r e a caused water intake in 30 % of infusions. Other workers have reported delayed or weak dipsogenic and antidiuretic effects of intracarotid hypertonic urea infusion 17,47. It is unlikely that these effects were due to rapid osmotic water withdrawal from osmoreceptors prior to diffusion of urea into these cells because of the relatively slow responses. Rather, the explanation of this phenomenon may be that sensor elements exist which are situated in, or influenced by the osmotic pressure of brain regions which possess a blood-brain barrier to urea. Since infusion of hypertonic urea should increase both sodium concentration and osmolality of CSF and interstitial fluid in such regions, such sensors could be responding to either change of Na concentration or osmolality. Turning now to the data obtained by making slow infusions of hypertonic artificial CSF solutions into the lateral ventricle, and summarised in Table V, it was observed that antidiuresis and consistent water drinking was caused by intraventricular infusion of hypertonic NaC1, sucrose, fructose but not urea - - all made in artificial CSF. In the case of the saccharides it is unnecessary to invoke a CSF sodium sensor in explanation of the responses, as CSF Na concentration should not have increased. As the more diffusible urea did not cause these responses the effects are consistent with an osmoreceptor and analogous to results in other species ~°,34,~. The relative lack of effect of intraventricular infusion of pure non-saline l M sucrose may have been due to the fact that CSF sodium concentration had been reduced as we have previously suggested 26, and confirms in this sense the importance of the CSF sodium
101 chloride concentration in the functioning of osmoregulatory responses 32,3a. In addition to reduction of NaC1 concentration of CSF, infusion of pure sucrose solution not made up in artificial CSF into the ventricle will also reduce the concentration of other important ions (e.g. K, Ca, Mg). However, pure 0.5 M NaCI caused rapid antidiuresis and thirst without correction of K, Ca and Mg concentrations, suggesting that the Na concentration is most important for these responses. While it may seem that the lack of antidiuretic and dipsogenic responses to intraventricular infusion of pure non-saline hypertonic solutions observed by the Swedish workers1,4,17, may be explained by failure to maintain basal CSF NaC1 concentrations, we observed that intraventricular infusion of hypertonic NaCI caused significantly greater water intake and more prolonged antidiuresis than the equiosmolar saccharide artificial CSF solutions when infused into the lateral ventricle. Thus, a specific effect of NaCI independent of its osmotic properties appears to be exerted. Initially, it was considered that this differential effect which we had previously observed on drinking behaviourz6, might be due to the differences in accessibility of stimulants to the receptors 34, or be due to non-specific neuronal influences because of the high NaCI concentration used relative to physiological levels. The evidence favours the alternative explanation that, in addition to osmoreceptors, there are also sodium or chloride specific sensors involved in control of water balance. If differential accessibility of the hypertonic agent to the receptors were involved, it could be expected that the latency of the responses would be different and this seemed not to occur. Rather, there was greater duration of the antidiuretic and dipsogenic effects caused by intraventricular hypertonic sodium solution. Non-specific neuronal excitation also seems less likely since, if this were the case, other behavioural effects (e.g. panting, general disturbance, hunger) could have been expected similar to those observed when KC1 is administered into the ventricle ~0. In addition, dilution by CSF and interstitial fluid will considerably reduce the initial high concentrations of the infusates. Blood pressure was elevated slightly by intraventricular infusion of hypertonic NaC1. but was unaffected by intraventricular infusion of 0.7 M sucrose CSF, suggesting that the antidiuretic effects of the intraventricular infusions were not dependent on a pressor response. In summary, the changes in CSF sodium concentration suggest that initial effects on water intake and urine production caused by intracarotid infusions of various hypertonic solutions are more consistent with mediation by brain osmoreceptors located outside the blood-brain barrier system than by periventricular sodium receptors. However, the greater dipsogenic and antidiuretic effects of intraventricular hypertonic NaC1 CSF infusion compared to intraventricular infusion of other equiosmolar saccharide CSF solutions suggests the participation of sodium (or chloride) sensors within the blood-brain barrier. We speculate that a dual sensing system in the brain involving osmoreceptors and sodium sensors may be involved in regulating water balance in sheep.
102 ACKNOWLEDGEMENTS This work was supported by grants from the N a t i o n a l Health a n d Medical Research Council of Australia, the Laura Bushell Trust, a n d the A u s t r a l i a n K i d n e y Foundation.
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