Blunted Cardiovascular Growth Induction During Prolonged Nitric Oxide Synthase Blockade
Abstract The goal of the present study was to characterize the activation profile of the growth-related enzyme ornithine decarboxylase (ODC) in cardiovascular tissue during hypertension induced by chronic NO synthase blockade in relation to the development of structurally based changes in the heart and blood vessels. In previously instrumented conscious rats, mean arterial pressure and ODC activation were measured in cardiovascular tissue of rats treated with Nω-nitro-l-arginine methyl ester (L-NAME; 100 mg/kg per day PO) for 4 hours and 1, 6, and 12 days. After 12 days of L-NAME treatment alone or in combination with 3% L-ornithine, structurally based hindlimb resistance properties were assessed. A marginal activation of ODC in the left ventricle and aorta was seen at 4 hours but returned to control levels at 1, 6, and 12 days of L-NAME treatment. A slightly prolonged yet transient activation of ODC occurred in the mesenteric vascular bed. Structurally based hindlimb vascular resistance was enhanced by 15% at maximum vasoconstrictor tone, and no change in cardiac mass occurred with L-NAME treatment. L-NAME+3% L-ornithine treatment resulted in a similar level of structural upregulation compared with L-NAME treatment alone. In summary, 12 days of L-NAME treatment resulted in only a modest change in vascular resistance, and only at maximum constriction, and no cardiac hypertrophy despite the presence of marked hypertension. The results of the present study indicate that either (1) pressure alone is not a sufficient stimulus to induce cardiovascular growth processes or (2) L-NAME may be “nonspecifically” inhibiting cardiovascular growth processes.
It is widely acknowledged that structural changes in the heart and blood vessels occur in almost all forms of hypertension. A less-defined association is the cause-effect relationship between the level of cardiovascular hypertrophy and the magnitude of the hypertension. The development of cardiovascular structural changes is a feature commonly associated with experimental models of hypertension such as two-kidney, one clip, SHR, or Ang II infusion.1 2 3 In contrast, in studies in which hypertension was induced by NO synthase blockade, this relationship does not hold.4 5 6 7 8
Studies by Arnal et al4 5 have demonstrated that increases in cardiac mass are normally lacking with hypertension induced by NO synthase blockade using the antagonist L-NAME. A small subgroup of these rats (25%), however, developed cardiac hypertrophy that was strongly correlated with increased plasma renin activity. On the other hand, in the rats that did not develop cardiac hypertrophy, the plasma renin activity was not elevated.4 5 The mechanism of the differential activation of the renin-angiotensin system has not been elucidated. Taken together, the findings indicate that in the majority of rats, during the first 2 to 3 weeks of NO synthase blockade the lack of development of cardiac hypertrophy is consistent with the absence of neurohumoral activation.4 5 6 7 8
Consistent with these findings with respect to the heart, the studies from Schiffrin’s group (Sventek et al9 ) as well as Dunn and Gardiner10 have both demonstrated that short-term L-NAME–induced hypertension leads to minimal9 or no change10 in vascular structure. Regardless of whether changes in structure occur, the magnitude of these changes was found to be inconsistent with the degree of hypertrophy found in other models of experimental hypertension.11 The reason for the lack of cardiovascular structural changes has not been elucidated. Schiffrin has proposed that L-NAME, despite inducing hypertension, may have growth-inhibitory properties independent of the effects on NO generation.11 12 Alternatively, Dunn and Gardiner10 have proposed that the lack of growth response is because increased pressure alone is not a potent enough trophic stimulus, which is consistent with the lack of activation of trophic neurohumoral systems.
Recently, studies by Banting et al13 and another group14 have revealed that the primary initiation mechanism of L-NAME hypertension results from the rapid upregulation of a powerful local vasoactive system, endothelin. ET has also been shown to be a potent inducer of cardiovascular growth both in vitro15 and in vivo, where Schiffrin et al16 have demonstrated its involvement in the development of vascular growth in the SHR DOCA-salt model of hypertension. Given that ET has been shown to have this growth-promoting capacity, the lack of evidence for trophic changes duringL-NAME hypertension is in conflict with the putative role of ET as a trophic factor.
To address some of the conflicts presented by these findings, we have determined the time course of activation of an obligatory growth-related enzyme, ODC.17 18 19 ODC is the first and rate-limiting step in the biosynthesis of polyamines, which are essential for protein synthesis, cellular proliferation, and tissue repair processes. Thus, activation of ODC is an essential step in the induction of cellular growth in all cells.17 18 19 To characterize the pattern of growth induction or lack thereof with NO synthase blockade, we assessed ODC activity levels throughout a 12-day treatment withL-NAME. In addition, we determined cardiovascular structural changes by assessing cardiac mass and structurally based vascular resistance properties in the hindlimb circulation after L-NAME treatment.
Male Sprague-Dawley rats (290 to 350 g; Charles River Laboratories, Montreal, Quebec, Canada) were housed individually under conditions of a 12-hour light/12-hour dark cycle at a room temperature of 22°C to 24°C; they were provided with Purina rodent chow and tap water ad libitum for at least 2 days before starting any procedures.
Measurement of ODC Activity in Blood Vessels and Heart
ODC activity in thoracic aortic (aortic arch to diaphragm), mesenteric vasculature (including vessels considered to be resistance vessels as well as the elastic and muscular segments of the superior mesenteric artery), and LV supernatant fractions was determined by the method of Russell and Synder20 (later modified21 ), in which 14CO2 released from DL-[L-14C] ornithine HCl was measured. Mesenteric and aortic tissues were homogenized in 10 vol and single LV in 5 vol of 10 mmol/L Tris buffer (pH 7.2), 0.5 mmol/L dithiothreitol, and 0.4 mmol/L pyridoxal-5′-phosphate. The homogenates were centrifuged at 13 000g at 4°C for 15 minutes. The reaction mixture contained 400 μL of supernatant, 2.0 μCi (96 μmol/L, aorta and mesentery) or 0.5 μCi (24 μmol/L, LV) of DL-[L-14C] ornithine HCl (specific activity, 42.5 mCi/mmol; New England Nuclear), 10 mmol/L Tris buffer (pH 7.2), 0.5 mmol/L dithiothreitol, and 0.4 mmol/L pyridoxal-5′-phosphate in a final volume of 0.5 mL. After incubation (60 minutes, 37°C) in a shaking water bath, HCl (100 μL, 3.0 N) was injected, and the mixture was shaken for another period (60 minutes, room temperature). Radioactivity in the CO2 trapping agent (Solvable, New England Nuclear) was counted. Blank values obtained from identical samples containing 5 mmol/L difluoromethyl-ornithine (Merrell Dow Pharmaceuticals Inc) were subtracted from determinations of each tissue supernatant. Protein concentrations of the supernatant were determined by the method of Lowry et al.22 The results of each ODC activity determination in treated rat tissues were compared with those of the control sample in that particular experiment. The data are expressed therefore as a fold difference relative to the control values.
Hindlimb Vascular Resistance Properties With NO Synthase Blockade
This procedure of comparing the perfusion of the isolated right hindlimb vasculature of rats with L-NAME for 12 days, alone or in combination with 3% L-ornithine–treated rats compared with control treated rats, is based on a technique established by Folkow et al23 and modified by Thompson and Adams.21 Control rats were treated acutely (≈30 minutes) with L-NAME (100 mg/kg IP) to control for the leftward shift of the cumulative MXA dose-response with NO synthase blockade.24 A heated box maintained both the temperature of the rats and the perfusion apparatus at 37°C to 38°C. The perfusion system consisted of a heated reservoir, an injection port, and a bubble/mixing chamber connected to a single peristaltic pump (Gilson, Minipuls 3). The perfusate was a Tyrode-dextran solution (1.5%; average mol wt, 71 200; Sigma Chemical Co) composed of (mg/100 mL fluid) KCl 20, CaCl2×H2O 32.3, MgCl2×6H2O 5.1, NaH2PO2×H2O 6.2, NaHCO3 100, glucose 100, and NaCl 800. The solution was maintained at pH 7.4, 37°C to 39°C, and oxygenated with 95% O2 and 5% CO2. The rats were anesthetized (60 mg/kg sodium pentobarbital) and heparinized (1000 IU/kg IV). After midline abdominal incision, the right iliac artery was cannulated proximal to the iliac bifurcation with a 21-gauge needle and connected to the perfusion apparatus. After the vena cava and spinal cord were sectioned to eliminate neural influences and to remove venous resistance, the exsanguinated rat was perfused at a constant flow rate (1 mL/min per 100 g body wt). The PP was continuously recorded on a data acquisition system (MacLab, ADInstruments). After time was allowed for the blood vessels to flush free of blood, sodium nitroprusside (20 ug/mL) was infused to produce maximum vasodilation13 25 based on a comparison to the maximal lowering of PP induced by papaverine.21 26 To ensure a common baseline condition, only preparations that had a stable PP at minimum vascular resistance at the end of a washout period were used. A flow rate–PP relationship was characterized by measuring the PP at minimum vascular resistance (PPmin) at flow rates of 0.5, 1, 2, and 4 mL/min per 100 g body wt. A cumulative concentration-response curve to MXA (0.5 to 64 ug/mL, 3 minutes per level) was generated until PP at maximum constriction with MXA was achieved. Subsequently, an infusion of supramaximal concentrations of constrictors (vasopressin, 10 IU/mL; Ang II, 200 ng/mL; MXA, 64 μg/mL) was given to ensure that a maximum constrictor response (PPmaxcon) was achieved that was not dependent on the activation of a single receptor type. After the concentration-response assessments were completed, flow was stopped to ensure that the pressure returned to zero.
The flow rate–PPmin relationships were plotted and the slope was calculated by linear regression analysis. The slope (PP/flow rate, mm Hg/[mL/min per 100 g body wt]) was used as an index of changes in lumen diameter integrated with vascular distensibility.22 23 The PP responses to MXA were computer fitted to a sigmoidal logistic curve (Sigmoid Version 5, Baker Medical Research Institute, Melbourne, Australia) to fit data points by the algorithm of least-squares estimates of nonlinear parameters.27 Values obtained directly from the collected data were PPmin and PPmaxcon (after bolus constrictor cocktail). The only values that were obtained from the fitted curves were EC50 and the maximum slope. The determination and comparison of these parameters were used as the best method to assess differences between treatment groups. At maximum dilation, the changes in PP reflect the hemodynamic consequences of changes in the average cross-sectional area of the vessel lumens.23 Any structural change in the vessel wall would alter the PP relationships. According to Poiseuille’s law, any change in the lumen radius averaged throughout the entire vascular bed would produce an inversely proportional change in resistance amplified to the fourth power. The PPmaxcon is used as a direct index of the contractile mass in the vascular bed. This characteristic has been shown to correlate directly with structural changes of increased wall thickness and media to lumen ratio.22 The EC50, the [MXA] that produced 50% of the maximal PP response, was used as an indication of the smooth muscle sensitivity to α1-adrenergic stimulation. Furthermore, an enhancement of the wall to lumen ratio of the vessels will increase the reactivity to constrictor agents whether it is the result of lumen and/or wall changes.23
Measurement of MAP in Conscious Rats
The surgical method for the implantation of catheters was based on the technique described by Head and Adams.28 Rats were anesthetized with ketamine/xylazine (70/5 mg/kg IP), and the descending distal aorta and inferior vena cava to the kidneys were catheterized with small-bore Teflon tubing (0.012-in inner diameter, 30 gauge outer diameter; Cole-Parmer). The catheter was filled with heparinized saline (10 IU/mL) and held in place by a small drop of cyanoacrylate tissue glue at the puncture site. The catheters were tunneled subcutaneously and exteriorized at the back of the neck and sutured in place. Two days after surgery, MAP was recorded (Narco Physiograph or MacLab DAS, ADInstruments). After connection, an equilibration period of approximately 30 minutes was allowed. Subsequently, a 5-minute average MAP was taken from each rat at 15-minute intervals for at least 1 hour (control baseline period) before the treatment period.
Time-Course NO Synthase Blockade
Acute NO synthase blockade (4 hours) was produced by a single injection of L-NAME (100 mg/kg IP). Chronic NO synthase blockade (longer than 4 hours) was produced by an initial injection of L-NAME (100 mg/kg IP) followed by 100 mg/kg per day in drinking water. The ODC activity after NO synthase blockade was assessed at 4 hours and 1, 6, and 12 days (n=8, 8, 5, and 7, respectively). LV to body weight ratios were calculated at all time points as an index of cardiac structural alterations. In a separate group of rats, hindlimb vascular resistance properties were assessed in controls (n=6) and after 12 days of NO synthase blockade alone (n=6) or with 3% L-ornithine (n=6).
All values are expressed as group mean±SD or SEM as indicated. Student’s unpaired t test with the Bonferroni correction method was used for statistical comparisons between groups. A value of P<.05 was considered significant.
MAP With NO Synthase Blockade
There was a 35-mm Hg increase in MAP (Fig 1⇓) observed within 5 minutes after L-NAME administration, an elevation that persisted throughout the 12 days of treatment. 3% L-ornithine treatment in the tap water did not alter the MAP profile in a separate group of L-NAME–treated rats (unpublished observations, K.E. Thompson, P. Friberg, and M.A. Adams, 1997).
Time Course of ODC Activation With Chronic NO Synthase Blockade
The time course of activation of ODC in the LV (Fig 2⇓, top) throughout the L-NAME treatment revealed only a brief, short-lived increase. After 4 hours of NO synthase blockade, ODC activity increased to 492±75 pmol of 14CO2/mg protein per hour above control levels (116±22 pmol of 14CO2/mg protein per hour), returning to control levels at 1, 6, and 12 days.
The aortic ODC activation profile paralleled the LV ODC activation after NO synthase blockade (Fig 2⇑, middle). After 4 hours of NO synthase blockade, aortic ODC activation was 1063±255 compared with control (571±53.8 pmol of 14CO2/mg protein per hour), returning to control levels at 1, 6, and 12 days.
The mesenteric ODC activation profile (Fig 2⇑, bottom) with chronic L-NAME treatment differed from the profile in the LV and aorta. ODC activation in the mesentery did not return to control levels on day 1, remaining significantly increased (893±322 versus control value of 364±77 pmol of 14CO2/mg protein per hour) but returning to control levels at 6 and 12 days.
Chronic NO synthase blockade did not induce an increase in cardiac mass. LV to body weight ratio determinations made at 4 hours (1.91±0.159 g/kg), 1 day (2.1±0.110 g/kg), 6 days (2.15±0.091 g/kg), and 12 days (1.89±0.095 g/kg) were not elevated above saline control values (1.97±0.27 g/kg).
Hindlimb Vascular Resistance Properties After 12-Day L-NAME Treatment
Analysis of the hindlimb vasculature revealed that only modest changes in resistance properties, only at maximum constriction, were induced by chronic NO synthase blockade. Chronic L-NAME treatment did not alter the PPmin at flow rates (mL/min per 100 g body wt) of 0.5 (14±2 versus 15±1 mm Hg), 1 (21±2 versus 21±2 mm Hg), 2 (30±3 versus 29±3 mm Hg), and 4 (44±5 versus 45±4 mm Hg) compared with control, respectively (Fig 3⇓). A logistic function analysis of the cumulative log [MXA]-PP response curves demonstrated that there was also no change in the sensitivity of the hindlimb vasculature after the treatment, as indicated by similar EC50 and maximum gain values compared with those of saline control.
On the other hand, the PPmaxcon obtained after bolus administration of MXA, vasopressin, and Ang II showed that there were significant increases (P<.05) in maximum resistance in the L-NAME–treated group (Fig 4⇓, bottom). Specifically, the PPmaxcon was increased by 15% in the L-NAME– and L-NAME+3% L-ornithine–treated groups compared with saline control (310±20 and 308±15 versus 270±19 mm Hg, respectively). The increase in maximum vascular resistance after a cocktail of vasoconstrictor agents has been previously demonstrated to be associated with an increase in “bulk” of the medial smooth muscle layer.23
The major findings of the present study include the following: (1) Despite the prolonged hypertension induced by L-NAME treatment, there were no changes in LV mass and only minimal changes in vascular resistance properties, which is consistent with (2) the observation that there was only transient activation of the obligatory growth-related enzyme ODC in the LV, the aorta, and the mesenteric vasculature, and (3) the supplementation with the ODC substrate L-ornithine (3%) did not alter the effect of L-NAME hypertension on cardiovascular structural changes.
The finding of both minimal cardiac and vascular structurally based changes during prolonged hypertension with L-NAME, although consistent with the previous data,4 5 9 10 is in contrast to the findings in almost every other model of hypertension. Thus, the “expected” result based on the level of hypertension induced by L-NAME would have been approximately a 35% upregulation of structurally based vascular resistance. This result was not achieved. Using the hindlimb perfusion technique, we determined that there was an “uncoupling” between the consequences to pressure and changes in vascular resistance properties (ie, there was no change in resistance at maximum dilatation). This perfusion technique, particularly under conditions of maximum dilatation, takes advantage of Poiseuille’s law and involves the assessment of structurally based resistance properties in all orders of vessels. This method provides a sensitive index of the hemodynamic impact of even very small changes in vascular dimensions according to the fourth-power relation with resistance. Our results confirm and extend previous findings regarding the lack of vascular structural changes during L-NAME treatment. Our novel findings in the intact vascular bed demonstrate that the minimal vascular changes are likely similar throughout the entire vascular bed.
It has been widely acknowledged previously that in different forms of experimental hypertension, “slow pressor mechanisms” (often distinct from the “initiating” cause) can account for long-term blood pressure elevation.29 Thus, in established hypertension, in almost all cases the “slow pressor mechanisms” have involved a critical role of vascular structural changes. In the last few years, it has become apparent that the quantitative contribution that these structural alterations make is greatest in the chronic steady-state phase of hypertension, at a time when the contribution of the basic initiating cause or stimulus29 has returned toward normal levels. This was previously explained by Folkow and coworkers1 23 as being part of a positive feedback loop in which the response to hyperactivity of a pressor mechanism is amplified by the development of pressure-dependent vascular hypertrophy, slowly leading to a substantial level of hypertension. Alternatively, others2 21 have proposed mechanisms that involve direct effects of trophic factors (eg, growth factors, Ang II, catecholamines) on the induction of cardiovascular growth processes. Regardless of the underlying mechanism, the consequences of these processes are increased structurally based vascular resistance and elevated blood pressure. In the present study, the results clearly do not support the concept that elevated blood pressure alone will necessarily result in proportional changes in cardiovascular structure. Previous results have demonstrated that in general there is a lack of activation of neurohumoral systems13 30 with L-NAME–induced hypertension. Hence, in the present study the incomplete structural adaptation to chronic hypertension may be causally related to the lack of prolonged activation of trophic systems.
Recently, Banting et al13 demonstrated a role of ET-1–mediated vasoconstriction as the initiating mechanism of acute L-NAME hypertension. Thus, the abrupt removal of the inhibitory actions of NO (by NO synthase inhibition) resulted in a rapid upregulation of ET vasoconstrictor actions. In the context of the present study, the specific role played by ET in the chronic model of L-NAME hypertension has not been elucidated. However, on the basis of previous data demonstrating that ET has the capacity to be a cardiovascular trophic factor, it may be that this mechanism plays a role in the minimal vascular structural changes found in the present study. Recent findings in DOCA-salt hypertension (in particular from Schiffrin’s group16 ) have supported the concept of a trophic role for endogenous ET in the development of vascular structural changes. They found that there is both increased ET-1 gene expression and immunoreactivity in blood vessels, but not in plasma, of DOCA-salt hypertensive rats. They further showed that the development of vascular hypertrophy in the DOCA-salt model was markedly attenuated by treatment with an ETA/ETB receptor antagonist. Schiffrin’s group has also demonstrated that L-NAME+DOCA-salt resulted in a blunting of the structural changes compared with DOCA-salt treatment alone.12 The vascular trophic capacity of ET-1 has also been supported by findings in cultured vascular smooth muscle cells, showing that addition of ET produces a mitogenic response.15 ET-1 is approximately 100 times more potent as a vasoconstrictor than Ang II or catecholamines. However, in the culture studies the maximal growth response to ET-1 was less than half that for Ang II. We are not aware of any studies that have assessed the in vivo cardiovascular growth responses to chronic ET infusion.
In the present study, the profile of the ODC activation was markedly blunted when compared with a similar time course of hypertension using equipressor levels of Ang II (unpublished observations, K.E. Thompson, P. Friberg, and M.A. Adams, 1997). We have determined that Ang II infusion unequivocally induces a proportional hypertension and upregulation of cardiovascular structure (unpublished observations, K.E. Thompson, P. Friberg, and M.A. Adams, 1997). The activation of ODC occurs before increases in mass in any tissue, and the duration of the increased ODC activity represents the time course of the growth phase.17 18 19 ODC is highly regulated in mammalian cells, which provides polyamine levels correlating with the cellular growth rate.31 Not surprisingly, pharmacological blockade of ODC inhibits cardiovascular growth responses in various experimental conditions.17 18 32 33 Thus, activation of ODC indicates a state of elevated cellular growth processes, whereas a return to basal ODC activity denotes a return to a “new” quiescent steady state. The lack of any growth signal at 6 days in any tissue suggests that the growth response follows an on-off trophic stimulus rather than a slow progressive time course.
Overall, the present study clearly indicates that rapid and sustained hypertension induced by NO synthase blockade is not a sufficient stimulus to induce a persistent activation of the growth-related enzyme ODC. These results indicate that either (1) pressure alone is not a potent trophic stimulus or (2) the NO synthase inhibitor L-NAME may be “nonspecifically” blocking cardiovascular growth processes, as has been proposed by Schiffrin11 and Li et al.12 Our data demonstrate that 3% L-ornithine supplementation neither augmented nor inhibited the L-NAME–induced vascular structural upregulation, suggesting at least that alterations in the ODC-polyamine pathway are not a likely mechanism for the lack of growth induction. Despite this latter finding, further work is required to elucidate the specific mechanisms involved in the lack of marked structural adaptation with chronic NO synthase blockade. We further speculate that an important component of the blunted development of cardiovascular structural changes is that there has not been a prolonged activation of neurohumoral systems acting as trophic factors.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|ET, ET-1||=||endothelin, endothelin-1|
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
|LV||=||left ventricle, left ventricular|
|MAP||=||mean arterial pressure|
|SHR||=||spontaneously hypertensive rat(s)|
This study was supported by research and traineeship (K.E.T., J.D.B.) funding from the Heart and Stroke Foundation of Ontario and Canada. M.A.A. holds a Career Award in Medicine from the Medical Research Council of Canada, the Health Research Foundation, and the Pharmaceutical Manufacturers Association of Canada.
Reprint requests to Michael A. Adams, Department of Pharmacology and Toxicology, Queen’s University, Kingston, Ontario, Canada K7 L 3N6.
- Received November 26, 1996.
- Revision received December 17, 1996.
- Accepted February 14, 1997.
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