Sympathoexcitatory Effect of Hypothalamic/ Hypophysary Inhibitory Factor in Rats
Abstract We recorded changes in arterial blood pressure, heart rate, and renal sympathetic nerve activity in response to intracerebroventricular injection of bovine hypothalamic/hypophysary inhibitory factor and ouabain in conscious Wistar rats. Ouabain at 0.3 to 0.6 μg caused dose-related increases in blood pressure, heart rate, and nerve activity (peak increases: 19±2 mm Hg, 42±4 beats per minute, and 48±4%, respectively; P<.05 versus basal). These responses were all blocked by central antibody Fab fragments, which bind ouabain and related steroids with high affinity. The inhibitory factor significantly increased blood pressure but decreased heart rate and nerve activity. Dose-dependent increases in blood pressure as well as heart rate and nerve activity were observed when the inhibitory factor was injected after intravenous injection of the vasopressin antagonist d-(CH2)5Tyr-(Me)AVP. Central Fab fragments, however, did not affect these responses. Both ouabain and the inhibitory factor inhibited Na+,K+-ATPase activity in vitro. Fab fragments blocked this inhibition by ouabain but not by the inhibitory factor. These data indicate that the ouabainlike sympathoexcitatory effect of this factor is masked probably by a potent central effect on vasopressin release. In contrast to rat brain “ouabain,” this factor does not exhibit a high affinity for the Fab fragments, supporting the previous finding that this compound is structurally a nonouabain Na+,K+-ATPase inhibitor.
- Na+,K+-exchanging ATPase
- hypothalamo-hypophyseal system
- sympathetic nerve activity
- immunoglobulins, Fab
The presence of endogenous Na+,K+-ATPase inhibitors in humans and experimental animals has been well established. These inhibitors are commonly described as digitalis-like or ouabainlike substances. Studies by Hamlyn et al,1 Mathews et al,2 and Tymiak et al3 characterized an endogenous Na+,K+-ATPase inhibitor in humans and animals structurally as ouabain1 2 or a closely related isomer.3 This endogenous ouabainlike substance (“ouabain”) may be synthesized in the adrenal cortex.4 “Ouabain” that appears not to be of adrenal origin5 is also found in high concentrations in rat brain6 and may actually be synthesized by mammalian hypothalamus.7 Several recent studies show an important role of brain “ouabain” in the development of sympathetic hyperactivity and salt-sensitive hypertension in spontaneously hypertensive and Dahl salt-sensitive rats8 9 as well as in sympathetic hyperactivity in congestive heart failure.10
Different from ouabain and ouabainlike compounds, a nonpeptide, nonlipid Na+,K+-ATPase inhibitor with low molecular weight has been isolated and purified from bovine hypothalamic and pituitary tissues.11 12 This compound, named HHIF, inhibits in a dose-related manner Na+,K+-ATPase and displaces ouabain from its receptors at the enzyme structure.11 12 It does not have ouabainlike cross-reactivity11 12 and inhibits the calcium pump of the synaptosome plasma membrane probably via disruption of the lipid annulus.13 So far, it has not been examined whether in vivo HHIF administered centrally may cause sympathoexcitation and hypertension, effects that have been shown for other brain or plasma-borne Na+,K+-ATPase inhibitors.14 15
In the present study, we (1) compared in conscious normotensive rats the changes in MAP, HR, and RSNA in response to acute ICV injection of HHIF, with or without pretreatment with intravenous AVP-ant, and compared these responses with those to ICV ouabain; (2) tested whether these responses can be blocked by ICV antibody Fab fragments, which bind ouabain,10 “ouabain,”10 16 and related steroids17 18 with high affinity; and (3) evaluated in vitro the effects of HHIF and ouabain on Na+,K+-ATPase activity with or without Fab fragments.
Male Wistar rats (175 to 200 g, Charles River, Montreal, Canada) were housed individually in rooms having a 12-hour light/dark cycle and room temperature of 23±2°C. All rats received standard laboratory rat chow and tap water ad libitum. All surgical and experimental procedures were carried out in accordance with the guidelines of the University of Ottawa Animal Care Committee for the use and care of laboratory animals.
After rats had 3 days for adaptation and while they were under sodium pentobarbital anesthesia, a 23-gauge guide needle was fixed with acrylic cement on the skull over the left lateral cerebral ventricle as a guide cannula (0.4 mm posterior and 1.4 mm lateral to the bregma). The lower end was 0.5 mm above the ventricle. At least 1 week later, rats were anesthetized with halothane inhalation and catheters were placed in the right femoral artery and vein. With additional intravenous methohexital sodium (30 mg/kg Brevital supplemented with 10 mg/kg as needed, Eli Lilly Canada Inc), a pair of platinum electrodes was placed around the left renal nerve through a left flank incision.15 After rats had recovered from the anesthesia (4 to 5 hours), they were placed in the original cage without restriction of movement. The intra-arterial catheter was connected to a pressure transducer, and BP and HR were recorded with a polygraph (model 7E, Grass Instrument Co) and tachograph (Grass 7P44). The electrodes were connected to a Grass P511 band-pass amplifier, and nerve activity (spikes per second) was counted by a nerve traffic analyzer (model 706C, University of Iowa Bioengineering). The background noise was recorded when the rats were killed at the end of experiments,15 and the actual RSNA was determined by subtracting noise from total activity. For ICV injection, a 26-gauge stainless steel needle was inserted into the guide cannula so that its tip protruded 0.8 to 1.0 mm from the tip of the guide cannula and was located inside the lateral ventricle. A 10-μL volume microsyringe (Hamilton) was used for ICV injection. All the compounds used in the experiments except Fab fragments were purchased from Sigma Chemical Co.
Rats were allowed to rest for 30 minutes after lines were connected. After the resting BP, HR, and RSNA had been measured, ouabain (1 μg per 0.5 mL for 10 seconds) and HHIF (25 U per 0.5 mL for 10 seconds) were injected intravenously at a 10-minute interval. The rats were thereafter randomly divided into four groups. In group 1 (n=6), ouabain at 0.3 and 0.6 μg per 1 to 2 μL was injected ICV. The next dose was given after the responses to the first injection had subsided for 5 minutes. Rats rested for 1 hour after disappearance of the responses, and 0.6 μg ouabain ICV was then repeated 5 minutes after ICV injection of Fab fragments (66 μg per 4 μL, Digibind). In group 2 (n=4), the same injections as in group 1 were repeated, with γ-globulins (66 μg per 4 μL) used as a substitute for Fab fragments. In group 3 (n=4), the same protocol as in group 1 was adopted, with ICV HHIF (2, 5, 7.5, and 25.0 U dissolved in 1 to 3 μL normal saline) substituted for ICV ouabain. Finally, in group 4 (n=10), the same injections of HHIF as for group 3 were performed, except that 5 minutes before each HHIF injection, the vasopressin V1 receptor antagonist [β-mercapto-β,β-cyclopentamethylenepropionyl1,O-Me-Tyr2,Arg8]-vasopressin, termed AVP-ant (Sigma Chemical Co),19 was injected intravenously to exclude the vasoconstrictor effects of possibly increased endogenous vasopressin.
HHIF was extracted from bovine hypothalamus and hypophysis. The method of successive chromatography for HHIF purification has been described previously.11 The quantity of HHIF was determined by measurement of its ability to inhibit Na+,K+-ATPase. One unit was defined as the amount of HHIF that inhibits 50% activity of 8 μg (7.2×10−11 mol) 90% purified Na+,K+-ATPase.11 The enzyme was purified from porcine kidney outer medulla.11
The in vitro assay for ouabain and HHIF for estimation of the inhibitory effect on Na+,K+-ATPase has been described previously.15 Quantification was done by measuring 32P liberation from [γ-32P]ATP (New England Nuclear) that was hydrolyzed by ouabain-sensitive Na+,K+-ATPase prepared from dog kidney (Sigma) with or without the presence of specific amounts of ouabain, HHIF, and Fab fragments.
Statistically significant differences between the responses before and after ICV pretreatment with Fab fragments or γ-globulins were determined by paired t test. ANOVA was used for other comparisons, followed by Duncan’s multiple range test. The level of significance was set at a value of P<.05.
Table 1⇓ summarizes the baseline values of BP, HR, and RSNA of the four rat groups used in the present study. No significant differences between groups were noted.
Responses to ICV Ouabain
Intravenous injection of 1 μg ouabain did not affect baseline BP, HR, or RSNA (data not shown). ICV ouabain at 0.3 and 0.6 μg caused dose-dependent increases in BP, HR, and RSNA (Figs 1⇓ and 2⇓). The responses started about 1 minute after ouabain injection, reached their plateau within 5 minutes (Fig 1⇓), and disappeared 10 to 35 minutes after the injection. Pretreatment with 66 μg Fab fragments or γ-globulins ICV did not change baseline values (data not shown, see Reference 1515 ). ICV Fab fragments nearly abolished the MAP, RSNA, and HR responses to 0.6 μg ouabain ICV (Fig 2⇓). ICV γ-globulins did not affect the responses to ouabain (Fig 2⇓).
Responses to ICV HHIF Without AVP-ant
No changes in MAP, HR, or RSNA were observed after intravenous injection of 25 U HHIF (data not shown). MAP started to rise in 10 to 20 seconds after ICV HHIF at each of the three doses, reached a peak level, and then fell to a level about two thirds of the peak within 2 minutes after the injection (Fig 1⇑). MAP then stayed at this level for about 20 to 30 minutes. In contrast, HR and RSNA decreased significantly 10 to 30 seconds after the ICV HHIF injection and gradually returned to resting levels about 1 to 10 minutes after the injection (Fig 1⇑), depending on the doses applied. Pretreatment with 66 μg Fab fragments ICV did not affect the responses to 25 U HHIF ICV. The peak changes in MAP, HR, and RSNA without and with Fab fragments are shown in Table 2⇓.
Responses to ICV HHIF With AVP-ant
Intravenous injection of AVP-ant at 30 μg/kg caused transient, minor (P=NS) decreases in BP, HR, and RSNA (Fig 1⇑). In contrast to the responses without pretreatment, after intravenous AVP-ant, ICV HHIF caused parallel increases in MAP, HR, and RSNA (Fig 1⇑). The increases reached a plateau 2 to 5 minutes after the injections (Fig 1⇑) and lasted about 10 to 20 minutes. The responses were dose-related (Fig 3⇓). ICV 66 μg Fab fragments had no effect on MAP, HR, or RSNA responses to 25 U HHIF ICV (Fig 3⇓).
In Vitro Na+,K+-ATPase Inhibitory Activity
Table 3⇓ shows two standard curves of Na+,K+-ATPase inhibition by ouabain and HHIF and the effects of Fab fragments on the inhibition of the enzyme. Both compounds exhibited a dose-related inhibitory activity. The inhibitory activity of 1 U HHIF was roughly equal to that of 30 ng ouabain (decreased enzyme activity to 76.3% versus 77.8% of controls). Fab fragments prevented the inhibitory effects of ouabain in a dose-related manner (6 μg Fab fragments blocked approximately 40 ng ouabain). In contrast, Fab fragments, even at a dose of 100 μg, did not affect the inhibitory effect of HHIF.
Similar to ouabain and “ouabain,”6 in the present study, HHIF inhibited Na+,K+-ATPase in vitro in a dose-related manner. In conscious rats, ICV HHIF alone caused a pressor response associated with sympathoinhibition and bradycardia but induced parallel sympathoexcitatory and pressor responses in a dose-related manner after intravenous AVP-ant. Pretreatment with antibody Fab fragments did not block these effects of HHIF in vitro or in vivo, unlike ouabain and “ouabain.”6 15
Since the isolation and purification of HHIF several years ago, several studies in vitro have been conducted to evaluate the actual effects of this compound. Despite their different chemical structures,11 12 HHIF and ouabain have similar effects.20 21 22 23 Consistent with previous studies,10 11 the present study shows that HHIF and ouabain inhibit Na+,K+-ATPase in a dose-related manner. One unit HHIF inhibits Na+,K+-ATPase activity to the same extent as 30 ng ouabain. This is comparable to the results in vivo showing that peak responses to 25 U HHIF are similar to the responses to 0.6 μg ouabain.
The present study is the first demonstration of the biological activity of HHIF in vivo. In conscious rats, centrally administered HHIF induced a significant increase in BP but significant decreases in HR and RSNA. After intravenous AVP-ant, however, ICV HHIF caused parallel increases in BP, HR, and RSNA in a dose-related fashion that were very similar to those induced by ICV ouabain and brain “ouabain.”15 In addition to its potent pressor effect, vasopressin also increases the sensitivity of the arterial baroreflex,24 25 possibly via a central action,26 and causes a greater increase in cardiac vagal efferent activity and decrease in sympathetic outflow in response to the increase in BP. Vasopressin also has a direct cardiodepressant effect to lower HR.27 Thus, intravenous vasopressin causes sympathoinhibition and bradycardia. The present study suggests that centrally administered HHIF can cause a pressor response by two mechanisms: HHIF appears to cause sympathoexcitation and hypertension, as does ouabain, but also appears to have a potent effect on vasopressin release; responses to the latter mask the sympathoexcitatory effects of HHIF. Similar to previous studies,8 9 28 ICV ouabain alone at the doses used in the present study caused only increases in HR and RSNA. Moreover, the sympathoexcitatory and pressor responses to ICV ouabain with and without AVP-ant are similar.8 14 28 Thus, it appears unlikely that ouabain, at least at the doses used, markedly increases vasopressin release. It is possible that after ICV injection, only compounds with a structure like HHIF but not ouabain can reach and inhibit the Na+,K+-ATPase in areas related to vasopressin synthesis or release.
Fab fragments did not affect the responses to central HHIF and its inhibitory effects on Na+,K+-ATPase activity in vitro. Fab fragments are capable of binding with high-affinity analogues of digitalis glycosides, such as digoxin,17 digitoxin,17 ouabain,10 and human16 and rat10 “ouabain.” In vivo, Fab fragments may diffuse rapidly into the interstitial space and bind free glycosides with affinity higher than that for glycoside binding to the Na+,K+-ATPase,18 therefore preventing or reversing the responses to ouabain such as sympathoexcitation and hypertension.15 The failure of Fab fragments to block the effects of HHIF in vivo and in vitro confirms that HHIF as a sodium pump inhibitor is structurally different from ouabain and digitalis glycosides.
In summary, similar to ouabain and “ouabain,”6 in vitro HHIF inhibits the Na+,K+-ATPase activity in a dose-related manner; in conscious rats, ICV HHIF causes a sympathoexcitatory response, which, however, becomes apparent only after intravenous AVP-ant. Fab fragments block the effects of ouabain and “ouabain”6 15 but do not affect the responses to HHIF, which is in agreement with the previously proposed11 nonouabain structure of HHIF.
Selected Abbreviations and Acronyms
|AVP-ant||=||vasopressin antagonist d-(CH2)5Tyr-(Me)AVP|
|HHIF||=||hypothalamic/hypophysary inhibitory factor|
|MAP||=||mean arterial pressure|
|RSNA||=||renal sympathetic nerve activity|
This study was supported by operating grants from the Medical Research Council of Canada and by Research Grant FIS 94/0497. Frans H.H. Leenen is a Career Investigator of the Heart and Stroke Foundation of Ontario, Canada. Digibind was a generous gift from Glaxo Wellcome Inc, Toronto, Canada.
- Received July 5, 1996.
- Revision received August 15, 1996.
- Accepted December 5, 1996.
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