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(Hypertension. 1997;30:1493-1498.)
© 1997 American Heart Association, Inc.
Articles |
Hypothalamic Hypertensive Factor
An Inhibitor of Nitric Oxide Synthase Activity
From the Department of Biochemistry (H.R.M., A.T.E., M.P., J.R.T.), Imperial College, and the Departments of Chemical Pathology (J.A.-Z., S.M.H., S.M., H.E. de W.), Biochemistry (J. de B.), and Psychiatry (I.D., N.S.K.), Charing Cross & Westminster Medical School, London, UK.
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Abstract |
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Abstract Human and rat plasma and rat hypothalamus contain a cytochemically detectable substance, the concentration of which rises with an increase in salt intake. The plasma concentration of this material is also raised in essential hypertension and in the spontaneously hypertensive rat (SHR), the Milan hypertensive rat, and the reduced renal mass (RRM) hypertensive rat. In the normal rat, the greatest concentration is found in the hypothalamus of the SHR and the RRM hypertensive rat. The physicochemical characteristics of this cytochemically detectable hypothalamic hypertensive factor (HHF), including chromatographic behavior and molecular weight range, suggest that it may share features common to a substituted guanidine that is present in established nitric oxide synthase (NOS) inhibitors. It was therefore decided to determine the effect on NOS activity of the HHF obtained from mature SHR. The ability of HHF to inhibit NOS activity was studied on (1) NOS extracted from bovine aorta, rat brain, and human platelets by measuring the conversion of radiolabeled L-arginine to L-citrulline and (2) rat liver NOS measured indirectly with a cytochemical technique based on the stimulation of soluble guanylate cyclase activity in hepatocytes by NO. HHF showed a biphasic inhibitory action on platelet NOS activity that was greater with HHF obtained from SHR than from Wistar-Kyoto rats. HHF also had a biphasic inhibitory effect on hepatocyte NOS activity that was more potent when obtained from SHR. It is proposed that the increase in HHF, a novel form of NOS inhibitor that is elevated in SHR, may be involved in the rise in arterial pressure.
Key Words: • hypothalamic extract • nitric oxide synthase inhibitor • rats, inbred SHR
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Introduction |
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Two cytochemical techniques have revealed the presence of an unidentified substance in the urine, plasma, and hypothalamus that reversibly inhibits Na+,K+-ATPase activity in the proximal tubule of the guinea pig kidney with a maximal effect at 4 to 6 minutes and reversibly stimulates G6PD maximally at 2 minutes.1 The chromatographic properties of the substance on HPLC and electrophoresis preclude ouabain as a structural candidate,2 and it does not influence leukocyte sodium transport (Poston, 1983) in the concentrations available or displace ouabain from the surface of red cells (Clarkson, unpublished observations, 1983). The plasma concentration of the cytochemically detectable substance in normal humans and the rat rises with an increase in salt intake.3 4 It is also raised in essential hypertension5 6 and in the SHR,7 the Milan hypertensive rat,8 and the RRM hypertensive rat.9 In the normal rat, the greatest concentration of the substance is in the hypothalamus, where it rises substantially with a high salt intake.4 The highest concentrations are found in the hypothalamus of the SHR7 and the RRM hypertensive rat.9
Purification of hypothalamic extracts from the SHR hypothalamus (including thin-layer chromatography, electrophoresis, HPLC, and ion-exchange chromatography) indicates that the substance is of low molecular weight and is highly polar, carrying a net positive charge at a neutral pH.2 9 Overall, microchemical derivatization experiments and the physicochemical properties of this HHF have suggested that it contains a quaternary or quaternizable nitrogen. It was found that choline and a related structure, DMMI, were detected by the cytochemical assay and have similar but not identical properties to HHF.2 DMMI, however, was detected at relatively high concentrations only. The fall in HHF activity in the plasma and hypothalamus, which occurs in SHR after the prolonged intracerebroventricular infusion of hemicholinium (an inhibitor of high-affinity neuronal uptake of choline), is consistent with the suggestion that the substance is a choline derivative.10 Further consideration of the structure of DMMI has led to the conclusion that HHF may contain certain atomic features in common with substituted guanidines, which are established NOS inhibitors.11 Here we report that in two separate assay systems, HHF is an inhibitor of NOS.
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Methods |
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Extraction and Purification of Hypothalamic Extract
SHR and WKY (bred by Harlan UK Limited) 9 to 11 weeks old were fed a standard laboratory diet for 6 weeks and then decapitated; the hypothalamus was removed immediately as described by Bradbury et al12 and placed in acidified acetone at room temperature. The tissue was homogenized with a glass rod and placed on a rotary mixer at 37°C for 1 hour. The extracts were then centrifuged, and the acetone was dried under N2. The dried fraction was resuspended in acidified water (HCl, pH 3), and lipids were extracted with chloroform. The hypothalamic extract was placed on precoated silica gel thinlayer chromatography and subjected to electrophoresis at a constant voltage for 1 hour; the electrolyte contained 78 mL glacial acetic acid and 24.4 mL 90% (vol/vol) formic acid, increased to 1L with distilled water. The presence of HHF was determined by its ability to stimulate G6PD in the proximal tubule of the guinea pig kidney at 2 minutes.1 The biologically active silica gel electrophoretic material was placed on an HPLC column (Dupont C18) and eluted at 1 mL/min isocratically with 0.1% trifluoroacetic acid for 15 minutes and then on a linear gradient to 100% methyl alcohol for 50 minutes. In the elution profile, HHF was found as a single peak of activity1 at the beginning of the organic gradient. In some experiments, HHF was further purified by elution from a Dionex ion exchange column with methanesulfonic acid. The active fractions of HHF from 100 hypothalami were freeze-dried in batches and stored at -70°C. The experiments were performed on dilutions of these preparations.
Detection of NOS Inhibitory Activity
The capacity of HHF to inhibit NOS was investigated on (1) NOS
extracted from human platelets by measuring the conversion of
L-arginine to L-citrulline and (2) rat liver
NOS by a cytochemical technique. Bovine aorta and rat brain (cerebral
cortex and cerebellum) were also used as a source of NOS, the activity
of which was assayed as for platelets.
Assay of Human Platelet, Rat Brain, and Bovine
Endothelial Cell NOS Activity by Measurement of the
Conversion of L-Arginine to
L-Citrulline
Washed platelets were prepared from blood samples obtained
from healthy volunteers as described by Essali et al.13
Vascular endothelial cells were removed from bovine
aortas (obtained from a local slaughterhouse) by careful rubbing of the
intimal surface with a scalpel blade. Platelet, bovine aortic
endothelial cells, and normal rat cortical and
cerebellar NOS extracts were prepared by sonication in extraction
buffer consisting of 0.25 mol/L sucrose, 100 nmol/L Tris
(pH 7.4), 1 mmol/L dithiothreitol, 1 mmol/L
EDTA, 100 µg/mL PMSF, 10 µg/mL leupeptin, 10
µg/mL soybean trypsin inhibitor, and 2
µg/mL aprotinin. Assays were performed by the incubation of
platelets (50 µg protein) at 37°C for 15 minutes in a total
volume of 0.1 mL containing 12.5 mmol/L HEPES (pH 7.3) with
1.2 mmol/L MgCl2, 0.96 mmol/L
CaCl2, 60 mmol/L L-valine, 1.2
mmol/L L-citrulline, 0.024 mmol/L
L-arginine, 120 000 dpm of radiolabeled
L-arginine, and 0.12 mmol/L
ß-NADPH.14
Platelets possess both constitutive (endothelial) and inducible forms of NOS.15 Endothelial NOS activity was measured by following the conversion of radiolabeled L-arginine to L-citrulline in the presence or absence of the hypothalamic abstract or 1 mmol/L of the inhibitor L-NMMA. Platelet NOS activity was found to be 1.7±0.4 nmol/L of citrulline formed per minute per gram of protein from nine separate assays.16 Results are expressed as the percentage of NOS activity over an incubation period of 15 minutes and sensitive to complete inhibition by 1 mmol/L NMMA. Assays were carried out with three or more platelet extracts at each concentration of inhibitor.
Cytochemical Assay of NOS Activity in Hepatocytes
A new quantitative cytochemical method was used for measuring
NOS activity in the liver17 18 that is based on the
stimulation of soluble guanylate cyclase activity in
hepatocytes by NO.19 20
Liver or brain from normal WKY was cut into segments and frozen in n-hexane at -70°C. Unfixed sections were cut from these segments in a cryostat at -30°C. Sections were incubated in reaction medium containing 30% polypep 5115, 1 mmol/L NADPH, 5 mmol/L arginine, guanosine triphosphate 1 mmol/L in 0.2 mmol/L Tris buffer that contained sodium azide 2.4 mmol/L, and lead ammonium citrate complex 32 mg/mL at pH 7.4 for 30 minutes in the presence of dilutions of purified hypothalamic extract from one hypothalamus. Basal guanylate activity was measured after suppression of NOS activity by the procedure being repeated with the same reaction medium described above but without NADPH, arginine, or hypothalamic extract. The two soluble enzymes, guanylate cyclase and NOS, are retained within the section by the collagen polypeptide. When guanylate cyclase acts on GTP, the pyrophosphate liberated is trapped by a special hidden-metal capture reaction that does not react with GTP or cause enzyme inhibition.21 After incubation, the sections were washed in distilled water and blackened by immersion in ammonium polysulfide. The guanylate cyclase activity in the hepatocytes was measured by microdensitometry, midway between the hepatic and portal vein from duplicate sections. Each result is the mean±SEM of 20 microdensitometer readings and expressed as mean integrated extinction x100. The stimulation of guanylate cyclase activity by NO appears to be highly specific. It occurs in the presence of L-arginine and NADPH; no stimulation occurs in the presence of L-arginine and NADH or with the inactive isomers D-arginine and NADPH. The stimulated activity is inhibited by methyl arginine (3 mmol/L). Higher concentrations (6 mmol/L) cause an even greater decrease in activity that may be caused by the presence of NO formed from endogenous substrate.
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Results |
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Effect of Hypothalamic Extract on NOS Activity in the Aorta, Brain, and Platelets as Assessed by Measurement of the Conversion of L-Arginine to L-Citrulline
Hypothalamic extracts were tested at a range of dilutions on NOS activity in four tissue preparations: bovine aorta, rat cerebral cortex, rat cerebellum, and human platelets.7 Hypothalamic extract had no detectable effect on cortical, cerebellar, and bovine endothelial NOS activity (Table 1
). A biphasic inhibitory
effect of SHR and WKY hypothalamic extracts was detected only in the
human platelet preparations. This is shown for three separate batch
extractions. In 12 of 14 experiments carried out with known
biologically active hypothalamic extracts (according to their ability
to stimulate G6PD), positive inhibition of platelet NOS was
demonstrated.
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Hypothalamic extracts from SHR caused a significantly greater
inhibition of platelet NOS activity compared with extracts from WKY
(Figure, a, b, c). Inhibition was
detected at dilutions between 10-3 and
10-10 of extract equivalent to one
hypothalamus. Maximal inhibition >80% was observed in all three
extracts shown in the Figure, but the potency of the extract varied.
This probably reflects differences in purification procedures.
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Effect of Hypothalamic Extract on NOS Activity in
Hepatocytes as Assessed Cytochemically
Initial experiments with hepatocytes established that
the inhibition of NOS activity produced by three batches of SHR
hypothalamic extract was biphasic; this was in agreement with all the
other cytochemical assays.22 For example, in a typical
experiment, serial dilutions of extract from one hypothalamus at
10-2.4,
10-3.4,10-4.4, and
10-5.4 inhibited NOS activity by 29%, 31%,
43%, and 11%, respectively. In subsequent experiments, it was
established that maximal inhibition was at dilutions of
10-4.2 and 10-4.5 of
extract from one SHR hypothalamus. The effect on NOS activity of
10-4.5 dilutions of hypothalamic extract,
equivalent to one hypothalamus from SHR and WKY, was compared on serial
liver sections from 6 Wistar rats. The guanylate cyclase
activity in the presence of L-arginine and NADPH was higher
than the basal guanylate cyclase activity. The
guanylate cyclase activity in the presence of
L-arginine and NADPH and 10-4.5
dilution of hypothalamic extract equivalent to one SHR hypothalamus was
lower than the guanylate cyclase activity in the presence
of L-arginine and NADPH alone (Table 2). The SHR hypothalamic extracts
inhibited NOS activity by 80% to >100%, and the effect of the WKY
extracts was an inhibition of 0% to 33% (P<.01,
Mann-Whitney U test) (Table 2). Hypothalamic extracts at
dilutions of 10-2.5 to
10-5.2 of extract equivalent to one SHR
hypothalamus had no significant effect on cerebral and cerebellar NOS
activity as assessed by its effect on guanylate cyclase
activity in the presence of L-arginine and NADPH (Table 2).
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Discussion |
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These results demonstrate the presence of a new endogenous NOS inhibitor in the hypothalamus and its elevated activity in SHR. The inhibitory effect of HHF was biphasic on both platelet and liver NOS. The biphasic response with cytochemical assays is the usual experience. For example, in the two cytochemical assays that are used to detect the presence of HHF, its capacity to inhibit Na+,K+-ATPase and stimulate G6PD is biphasic. There are many well-documented examples of this biphasic reaction as illustrated by the cytochemical assays for parathyroid hormone, adrenocorticotropic hormone, thyroid-stimulating hormone, and vasopressin.22 That an inhibitory effect of HHF on liver NOS should be biphasic is therefore not surprising. It is the inhibitory effect of HHF on platelet NOS, as measured by the conversion of L-arginine to L-citrulline, that is striking. The variability in the inhibitory effect of different extracts of HHF was presumably caused by the described differences in purification procedures. Nevertheless, the pattern of inhibitory change found with the various batches was similar. When the effects of SHR and WKY HHF on hepatocyte NOS activity were compared, the cytochemical assays were carried out at the same time, with the same batch of extract, on the same platelets, and on slices of the same liver.
The physicochemical characteristics of purified hypothalamic material,
including the absence of 210 nm absorbance, permitted us to exclude its
identity from any endogenous NOS inhibitors
that have been isolated from the brain, including
NG-monomethyl,
NG,NG-dimethyl,
NG,N'G-dimethyl-L-arginine,
methyl lysine,23 24 and several other guanidino compounds,
which include 
-keto-
-guanidinovaleric acid, guanidinosuccinic
acid, creatine, guanidinoacetic acid, -N-acetylarginine,
argininic acid, ß-guanidinopropionic acid,
creatinine, 
-guanidinobutyric acid, arginine,
homoarginine, guanidine, and methylguanidine.11
Furthermore, whereas these inhibitors are nonspecific and
inhibit all known forms of NOS, HHF does not inhibit NOS from the
cerebellum, the cortex, or the aorta. Because HHF inhibits at very low
dilutions, it may be acting at the cofactor level in NOS inhibition, a
hypothesis that we are currently investigating.
The functional characteristic used to isolate HHF from the hypothalamus is its ability to stimulate G6PD, which has been demonstrated to correlate with its ability to inhibit Na+,K+-ATPase. It may be relevant therefore that ouabain, a specific Na+,K+-ATPase inhibitor, has been shown to inhibit the synthetic release of endothelial-derived relaxing factor (an L-NMA sensitive pathway) from human resistance arteries when stimulated by acetylcholine.25 At the concentrations of HHF used and with the particular methods used, however, it has not been possible to demonstrate an effect of HHF on NOS obtained from the aorta.
The rat hypothalamus contains a high concentration of NOS in the paraventricular and supraoptic nuclei.26 27 28 29 There is also a large cell group in the lateral hypothalamus, the processes of which form a dense network. The fornix, the subfornical organ, the laminal terminalis, the anterior and rostral periventricular areas, and certain nuclei in the ventromedial nucleus also contain various quantities of neurons with moderate to weak immunochemical staining for NOS.28 Brain NOS consists of neuronal NOS (90%) and endothelial NOS (10%).30 The only detectable endothelial NOS activity within or adjacent to the hypothalamus is in the supraoptic nucleus.31
The first intimation that NO might have a central effect on the blood
pressure arose from the finding that an intravenous bolus
of the NOS inhibitor L-NMA causes a rise in
arterial pressure accompanied by a paradoxical increase in
renal nerve activity32 and abolished by
hexamethonium.33 It was then demonstrated
that a bolus injection or a prolonged infusion of one of the
nonselective NOS inhibitors L-NMA, L-NAME, or L-NMMA
intracerebroventricularly in the normal
rat or the deoxycorticosterone acetate–salt rat causes a rise in
arterial pressure.34 35 36 The
arterial pressure also rises with the central
administration of Rp-8-Br cyclic GMP, a cyclic nucleotide
that blocks the action of cGMP (the effector product of the action
of NO on guanylate cyclase) on cGMP-dependent protein
kinase.35 On the other hand, the injection of an NO donor
(sodium nitroprusside) or calcium chloride (to activate NOS)
into the third ventricle or into the paraventricular nuclei
induces a fall in blood pressure.35 37 This was confirmed
to be due to NO by bilateral microinjection of the
paraventricular nuclei with 
32 pmol of NO in artificial
CSF for 30 minutes; this also caused a fall in blood
pressure.37 In more caudal areas of the brain, the effect
of NO is dependent on the site of administration. In the rostral
ventromedullary nucleus and nucleus solitarius, NO causes a fall in
blood pressure, and in the caudal ventromedullary nucleus it causes a
rise in blood pressure.38 39
There are two observations suggesting that there is a diminution of NOS activity in the SHR hypothalamus. The changes in arterial pressure induced by the central administration of the NOS activator calcium chloride and the NOS inhibitor L-NAME are significantly less in SHR,40 whereas the content of nitrite and nitrate in the SHR hypothalamus is less than in the hypothalamus of WKY.41
In contrast to nonselective NOS inhibitors, an acute intraperitoneal injection42 or a prolonged intracerebroventricular injection of a highly selective neuronal NOS inhibitor, 7-nitro indazole, into a normal rat has no effect on the blood pressure.43 Furthermore, the blood pressure of mutant mice that lack neuronal NOS is normal,30 with a tendency to hypotension when exposed to anesthesia; however, the blood pressure of mice in which the gene-encoding endothelial NOS is disrupted is hypertensive.44 These gene studies and those with NOS inhibitors suggest that the NOS isoform in the brain that is concerned with the blood pressure is not neuronal NOS and that it may be endothelial NOS.
HHF does not inhibit NOS obtained from rat cortex or cerebellum, but it does inhibit NOS obtained from human platelets and rat hepatocytes, in both of which NOS activity is considered to be due to endothelial NOS.45 Accordingly, HHF, by inhibiting hypothalamic endothelial NOS, may be directly involved in the pathogenesis and development of hypertension. The lack of effect of HHF on endothelial NOS obtained from bovine aorta suggests that HHF works through a different mechanism, which might be based on a specificity for another isoform of NOS or by tissue-specific modulation of NOS activity through the regulation of cofactor availability or other factors.
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Selected Abbreviations and Acronyms |
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-methyl-L-arginine
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Acknowledgments |
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This work was supported by a grant from the British Heart Foundation and the Trustees Research Committee at Charing Cross Hospital, London. We thank Dr Alan Barry Lansdown for his assistance.
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Footnotes |
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Reprint requests to Prof H.E. de Wardener, Department of Chemical Pathology, Charing Cross & Westminster Medical School, St Dunstan's Rd, London W6 8RP, UK.
Received June 11, 1997; first decision June 12, 1997; accepted June 12, 1997.
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