MATERIALS AND METHODS
Fifty-eight male SHRs (purchased from Elevage Janvier, France), were included in this study. This work was divided into 2 parts. The first one aimed to assess the most efficient way of systemic administration of a low dose of rilmenidine. In the second part, the optimal procedure of administration was used to investigate more precisely the effects of the chronic treatment with rilmenidine in this strain of rats. All methods employed in this work are in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publications No. 85-23, revised 1996).
Twenty-eight 11-week-old rats were used for this part of the study. Before the animals were included, their systolic blood pressure was measured with an indirect tail-cuff blood pressure system (Sphygomanometric device, Roucaire, France). Five animals that had a systolic blood pressure lower than 150 mm Hg were rejected. Rats were randomly divided into 3 groups. Animals of the first group (called “Control group”) were treated with intraperitoneal (i.p.) injections of saline twice a day (2 × 200 μL/d at 9 am and 6 pm) during 30 days. Rats of the second group (called “Discontinuous rilmenidine”) were treated with 2 intraperitoneal injections of rilmenidine per day (2 × 250 μg/kg/d at 9 am and 6 pm) during 30 days. In the last group (called “Continuous rilmenidine”), under a slight pentobarbital anesthesia (30 mg/kg i.p.) (Pentobarbital sodique, Sanofi, France), an osmotic minipump (Alzet 2002: total volume = 200 μL with an output = 0.5 μL/h; purchased from Charles River, France) was implanted in the abdominal peritoneal cavity. Rilmenidine was dissolved in sterile saline solution and its concentration in the pump was adjusted according to each animal's body weight to obtain a delivery of 500 μg/kg/d. After 15 days, the pump was replaced by a new one filled in the same conditions to obtain a treatment of 30 days. At the end of each period, all the pumps were checked and were nearly completely empty. No rats were implanted with osmotic minipumps delivering saline solution alone.
After 1 month of treatment, the cardiovascular parameters were recorded by the means of a heparinized (Heparine: 100 UI/ml of saline; Heparine Léo, France) polyethylene catheter (PE 20) inserted in the abdominal aorta via the right femoral artery. This catheter was implanted during a pentobarbital anesthesia (60 mg/kg i.p.), tunneled subcutaneously and exteriorized at the back of the neck. To measure the blood pressure parameters: systolic (SBP, mm Hg), diastolic (DBP, mm Hg), and mean (MBP = DBP + [1/3 SBP-DBP], mm Hg) and the heart rate (HR, beats/min), the tip of the catheter was connected to a Statham P23 Db transducer, which was in turn connected to a pressure processor and recorder (Gould Electronics model BS-272, Longjumeau, France).
All of the cardiovascular parameters were recorded during the pentobarbital anesthesia immediately at the end of the surgery, 30 minutes after the morning's injection of the treatment corresponding to the maximal efficacy of rilmenidine given by this route (for the 2 groups treated with intraperitoneal injections) or at the same morning time (ie, 9:30 am) for the “Continuous rilmenidine” group. The arterial pressure was continuously monitored during 20 minutes, after 10 minutes of stabilization. The given values of systolic, diastolic, and mean arterial pressures are the means of 4 values averaged in 5-minute blocks. The heart rate values are the means of 4 instantaneous measurements recorded every 5 minutes. Then, the rats were left to a spontaneous recovery and treatments with intraperitoneal injections were not disrupted. Measurements after the surgery and during pentobarbital anesthesia were performed after 29 days of treatment in both groups. This measurement was performed to reveal the antihypertensive action of the drug delivered continuously. All of these recordings were repeated 1 day later, at the same time (ie, 30 minutes after the last injection of the treatment or at 9:30 am for rats treated with Alzet mini-pumps) with the rats conscious and freely moving. The cardiovascular recordings were performed exactly as previously described.
Assessment of the Stability of Rilmenidine
In 12-week-old pentobarbital anesthetized SHRs, we compared the cardiovascular effects of rilmenidine (injected intravenously as a single bolus injection of 300 μg/kg) prepared freshly, to the ones of rilmenidine left 14 days at 37°C 6 in an incubator (in saline solution at the same concentration as fresh rilmenidine).
Radioligand Binding Studies
Rat cerebral cortex and kidney membranes were prepared as previously described 8 and stored at −80°C until use. Protein concentrations were determined by the method of Bradford (1979) with serum albumin as the standard.
Saturation experiments were performed by using 2 sets of triplicate tubes that contained 100 μL of membrane suspension (200 μg protein or 400 μg for brain and kidney respectively) and 20 μL of the appropriate concentration of [3H]rauwolscine (0.3, 1, 2, 4, 6, 8, 10, and 30 nM) in the presence of 1 μM serotonin (to block 5HT receptors that rauwolscine normally recognizes in addition to α2-adrenergic receptors) in a total volume of 400 μL. One set contained 10 μM phentolamine to determine nonspecific binding. Specific binding was calculated as the difference between total and nonspecific binding. After 45 minutes of incubation at 25°C, the suspensions were filtered through GF/B glass fiber filters (Whatmann) by using a 48-sample manifold (Brandel Cell Harvester). Tubes and filters were washed three times with 5 mL of ice-cold 50 mM Tris HCL (pH 7.4) and the radioactivity retained on the filters was determined by liquid scintillation spectroscopy. The Kd and Bmax values were calculated by a computer-assisted nonlinear regression of bound versus free ligand concentration. Results are given as mean ± SEM.
Eighteen rats were included in this part of the study. For the “Continuous” group, ALZET osmotic minipumps were implanted as previously described in 9 animals. A blood collection (500 μl) was performed on a heparinized syringe from a tail vein, in the conscious state, at 9 am on days 2, 5, and 7 of perfusion. Blood was immediately centrifuged at 2500 rpm during 10 minutes and the plasma was collected and stored at −20°C until use. For the “Discontinuous” group, 9 rats were intraperitoneally injected by 250 μg/kg of rilmenidine at 9 am and 6 pm every day during 7 days. Blood collections (500 μl) were performed at day 1 and day 7. At a given day, each rat was sampled 3 times with a matrix that allowed us to obtain 3 points of measurement at the following times after injection: 0.5, 1, 2, 3, 4, 6, 9, 10, and 24 hours. The samples of times 9 and 24 were performed just before the 6 pm and the 9 am (following day) injections, respectively. Blood and plasma were treated the same way for the “Continuous” group. Rilmenidine (S3341) was measured in plasma samples by solid phase extraction followed by LC separation and MS/MS detection. The method is linear from 0.5 to 100 ng.ml−1 and the limit of quantitation is validated at 0.5 ng.ml−1 using 100 μL of plasma. This analysis was carried out at Cephac Europe (Saint Benoit, France).
Animals and Hemodynamic Measurements
Twelve 10-week-old rats were randomly divided into 2 groups after their blood pressure was recorded. Under pentobarbital anesthesia, a 20-MHz soft silastic Doppler flow transducer of 1.6 mm of inner diameter (Ealing France for Harvard Biosciences, Les Ulis, France) was placed around the abdominal aorta (just beneath the renal arteries). Its cable was tunneled subcutaneously, plugged to an electric connector and fixed on the skull with dental cement (Paladur, Germany). The flow probe was connected to a flow processor (Triton Technology, San Diego, USA), which was in turn connected to an analogic acquisition data system (Clodia, Saint-Maur des Fossés, France). The relative variations of the flow values were extrapolated from the shift of the Doppler signal (calculated from the area under the speed flow curve). The variations of the hindquarter limb peripheral arterial resistances were calculated with the following equation:EQUATION
The quality of the signal was checked and the rats were left to a spontaneous recovery.
One week later, treatments began. Six rats were treated with saline (2 × 200 μL/d i.p. at 9 am and 6 pm during 30 days) and 6 animals were treated with rilmenidine (2 × 250 μg/kg/d i.p. at 9 am and 6 pm during 30 days). Before beginning the treatment (at J0) the systolic blood pressure and the heart rate (indirect tail cuff method), weight, and hindquarter blood flow were measured. Cardiovascular recordings were repeated every 10 days, at J10, J20, and J30, 30 minutes after the morning injection; the rats were conscious and immobilized in a restrainer. The rats were then killed by a lethal dose of pentobarbital (150 mg/kg i.p.), their hearts removed and fixed in a 4% formaldehyde solution, after the blood contained in the atrial and ventricular cavities was carefully drained.
Morphometric Evaluation of the Ventricular Mass
The analysis was performed after 2 weeks of fixation. Heart weight was measured (HW) (g) before cutting it in slices of 1-mm thickness from the atrio-ventricular groove to the apex. The third slice from the cardiac apex was examined with a computer-assisted planimetry (Image 1.44, NIH, USA), to determine 2 parameters: the left ventricular wall area (LVWA) (cm2) and the area of the left ventricular lumen surface (LVLS) (cm2).
All the results were expressed as mean ± SEM. Differences among groups were tested by one way or repeated analysis of variance (ANOVA) followed by the Scheffe test to detect statistically significant differences. A P value lower than 0.05 was considered statistically significant. The comparisons of pre- to post-treatment values in each group were compared using paired Student t tests. Moreover, when possible, values recorded at the end of the treatments in different groups were compared with one-way ANOVA followed by Bonferroni post tests. Here also, a P value lower than 0.05 was considered significant. All these calculations were made by computer-assisted analyses with the Stat-View II program (Abacus Concepts, Inc., Berkeley, USA).
There were no statistical differences in the systolic blood pressure and the heart rate recorded in the 3 groups of rats before the beginning of the treatments. These parameters were respectively 159 ± 3 mm Hg and 399 ± 5 beats/min in the “Control” group (n = 10), 159 ± 3 mm Hg and 395 ± 7 beats/min in the “Discontinuous rilmenidine” group (n = 9), and 154 ± 4 mm Hg and 407 ± 13 beats/min in the “Continuous rilmenidine” group (n = 9).
Blood Pressure and Heart Rate under Pentobarbital Anesthesia
In pentobarbital-anesthetized rats discontinuously treated with rilmenidine (2 × 250 μg/kg/d), SBP, DBP, MBP, and HR were lower than in saline-treated animals (Table 1). Mean blood pressure decreased by nearly 30% when the heart rate was reduced by 20%. At the opposite, continuous administration of rilmenidine (500 μg/kg/d) did not significantly modify blood pressure. In this case, only the heart rate was significantly reduced by 15% (P < 0.05). Moreover, when rilmenidine was intraperitoneally perfused, the blood pressure of the rats was significantly higher as compared with rats treated with 2 injections of the same daily dose.
Blood Pressure and Heart Rate of the Conscious and Freely Moving Rats
When the rats were conscious and freely moving, the discontinuous treatment with rilmenidine (2 × 250 μg/kg/d) persisted to be effective on the blood pressure but in a lesser extent than during pentobarbital anesthesia (−15%) (P < 0.05, n = 10 vs n = 9). This change in blood pressure was similar to the one observed in comparable SHRs receiving a first single injection of 250 μg/kg of rilmenidine. In this series, heart rate was not significantly reduced by about 13%. At the same time point of measurement, the continuous treatment (500 μg/kg/d) did not significantly change blood pressure (3% reduction) and heart rate (12% decrease) (Table 2).
Assessment of the Stability of Rilmenidine
The cardiovascular effects of rilmenidine, at a single bolus intravenous dose of 300 μg/kg, left 14 days at 37°C 6 in an incubator, in saline solution, were the same as those obtained with the same dose (at the same concentration) prepared freshly (control group). Systolic blood pressure decreased by 22 ± 1% in the first group versus 21 ± 2% in control rats, diastolic blood pressure diminished respectively by 21 ± 2% and 23 ± 1%, and heart rate fell respectively by 20 ± 2% and 15 ± 3% in control animals (P > 0.05, n = 4 in each group).
Radioligand Binding Studies
In saturation experiments performed with [3H]rauwolscine in renal membrane preparations from the 3 groups of animals, no significant modification of affinity and density of α2-adrenoreceptors was observed. In brain cortical membranes, Kd values were not significantly different among the 3 groups whereas Bmax of [3H]rauwolscine decreased by about 50% in rats treated discontinuously with rilmenidine compared with control animals. In rats treated with osmotic minipumps, a 400% increase of the Bmax of [3H] rauwolscine was observed (Table 3).
In the first day of treatment, 30 minutes after the first intraperitoneal injection of 250μg/kg of rilmenidine (n = 9), the plasma concentration was 32.2 ± 3.9 ng/ml. Then, 9 hours later, it rapidly decreased to 2.1 ± 0.8 ng/ml before the 6 pm administration. The residual concentration, 24 hours after the first injection and just before the third injection (9 am, day 2), was 1.1 ± 0.5 ng/ml. This kinetic profile was similar after 7 days of treatment, the curve being nearly superimposed to the one obtained the first day (P > 0.05, day 7 vs day 1) (Fig. 1A). When rilmenidine was perfused intraperitoneally, the plasma concentration varied from a minimal value of 8.4 ± 1.2 ng/ml (day 5) to a maximum of 12.8 ± 2.9 ng/ml (day 7). There was no significant difference among the 3 concentrations measured (P > 0.05, n = 9 on each day) and the mean plasma concentration obtained during this week of treatment was 11.1 ± 1.7 ng/ml (Fig. 1B). This last concentration is 3 times lower than the maximal concentration measured after a single 250 μg/kg i.p. administration but is 10 times higher than the residual value for the discontinuous schedule. In this last procedure, a concentration higher than 10 ng/ml can only be achieved for approximately 6 hours a day.
Comparison of the Animals at the Beginning of the Treatment Period
The two groups of rats were comparable for their systolic blood pressure, heart rate, and weight (one-way ANOVA for comparisons) (Table 4, D0). Concerning the blood flow and the hindquarter limb vascular resistances, a statistical comparison between the two groups was not performed because of the differences of blood flow values recorded from one probe to the other. As a matter of fact, the measurement of the flow with Doppler blood flow probes depends on the exact position of the probe on the vessel and so on the orientation of the incident pulsed signal. Therefore, only intra-group comparisons were performed for these two last parameters.
Effects of the One-Month Treatment with Rilmenidine in Conscious Spontaneous Hypertensive Rats
For the entire the treatment period, rilmenidine (2 × 250 μg/kg/d i.p.) lowered the systolic blood pressure; these effects were statistically significant at J10 and J20 (intra-group comparison) (Table 4). At J20, a 20% reduction of blood pressure was obtained as compared with the pre-treatment value (122 ± 5 vs 153 ± 4, P < 0.05). A statistically significant reduction of heart rate was noticed only after 10 days of treatment (342 ± 7 vs 409 ± 16, P < 0.05, n = 6 in each group) (Table 4). The hindquarter limb peripheral resistances decreased in rats treated with rilmenidine; the blood flow in these rats was unchanged. This effect reached a nonsignificant 29% reduction after 10 days (P = 0.06) and a 32% significant decrease after 20 days (P < 0.05 vs pretreatment).
Morphometric evaluation of the left ventricular mass
The treatment with rilmenidine did not modify any of the following cardiac morphometric indexes in control rats as compared with rilmenidine treated animals: heart weight (respectively 1.18 ± 0.09 g and 1.13 ± 0.04 g; P > 0.05), left ventricular wall area (respectively 0.74 ± 0.02 cm2 and 0.79 ± 0.03 cm2; P > 0.05), and surface of the left ventricular lumen (respectively 0.09 ± 0.06 cm2 and 0.05 ± 0.01 cm2; P > 0.05).
In the present study, a chronic (30 day) discontinuous treatment with 500 μg/kg/d of the centrally acting antihypertensive drug rilmenidine induced a sustained antihypertensive action in SHRs, when measured at the peak of the plasma concentration, which was not accompanied by any significant reduction of the cardiac hypertrophy. We also show that rilmenidine is very rapidly eliminated when delivered systemically in these animals.
Rilmenidine is known to cause a vasoconstriction through a direct vascular α-adrenergic stimulation. 8,9 Therefore, a long-term treatment with high doses of rilmenidine may increase the peripheric vascular resistances and thus counteract its central sympatho-inhibitory action. In addition, a chronic continuous infusion could induce a desensitization of the receptors involved in its effects. In preliminary experiments, the 500 μg/kg/d dose was selected as the least efficient one; 30-day treatment periods with doses of 50 and 100 μg/kg/d were ineffective (data not shown). When continuously delivered (500 μg/kg/d by the mean of osmotic Alzet® minipumps), rilmenidine was ineffective in animals under pentobarbital anesthesia as well as in conscious and freely moving animals. This lack of efficacy was not due to the degradation of rilmenidine in minipumps because this substance is stable in saline solution over 30 days at the rat's body temperature. Conversely, rilmenidine given discontinuously the same way (2 × 250 μg/kg) reduced blood pressure in anesthetized as well as in conscious animals. The blood-pressure lowering effect of rilmenidine was even greater in anesthetized rats. Such a potentiation of centrally acting hypotensive drugs by barbiturate anesthetics was previously described with clonidine and with rilmenidine. 1 Different hypotheses were proposed to explain this potentiation: a barbiturate-induced inactivation of some rostral vasopressive brain structures that are normally stimulated by clonidine-like drugs in conscious animals 10,11 or a pentobarbital-induced blockade of the counteracting baroreflex response evoked by the hypotensive drugs. 12,13 In our study, differences observed between “continuous” and “discontinuous” treatments were not dependent on the presence of an anesthetic agent as they were observed in awake animals as well as in anesthetized ones. Some authors suggested that in addition to its central sympathoinhibitory effects, rilmenidine could dilate microvessels through a stimulation of presynaptic α2-adrenergic receptors. 14,15 If this were true, the differences observed here could be due to the desensitization of the presynaptic α2–adrenergic receptors of neurovascular junction provoked by the chronic continuous infusion of the drug. To test this hypothesis, saturation experiments with the α2-adrenergic antagonist 3,4 rauwolscine were performed in kidneys of the treated rats. The evaluation of the desensitization of these receptors was performed in this tissue because it was known to contain the same α2b-subtype as the vascular wall. 16 In our experimental conditions, no desensitization occurred when the 2 groups of rats treated with rilmenidine were compared with control rats; neither Bmax nor Kd were significantly changed. Similar results were previously obtained in kidneys from rabbits chronically treated with rilmenidine. 17 Therefore, a peripheral desensitization process of α2-adrenoceptors cannot explain the differences that we observed in the present study. Central α2-adrenergic receptors (mainly of the 2a subtype) were also studied with [3H]-rauwolscine in the brain cortex. This radioligand was known to bind to all the α2-adrenergic receptor subtypes (ie, α2a, α2b and α2c). The various treatments did not significantly affect the Kd value of [3H]-rauwolscine bound to these receptors. At the opposite, the Bmax values were largely modified in rats treated with rilmenidine. In rats treated discontinuously, the Bmax for [3H]-rauwolscine decreased by nearly 50%. Despite this partial desensitization of the central α2-adrenergic receptors, no reduction of the antihypertensive efficiency of rilmenidine was observed, as clearly shown in the second part of our protocol (see following discussion). Nevertheless, it could explain, at least in part, the time-course of its bradycardic action (initially important and progressive decrease) that was observed in the second protocol. These data corroborate the hypothesis according to which the bradycardia induced by such drugs could be due to α2-adrenergic receptor stimulation. 18 Surprisingly, the Bmax values of [3H]-rauwolscine increased by nearly 400% in brains of SHRs treated with osmotic minipumps. At the present time, we cannot explain this important overexpression of α2-adrenergic receptors and we do not know whether this phenomenon could account for the lack of blood pressure effect of rilmenidine continuously delivered.
The kinetic course of the cardiovascular effects of 2 daily intraperitoneal injections of 250 μg/kg of rilmenidine during 30 days was investigated. In addition to the blood pressure and the heart rate effects, the hindquarter blood flow resistances and the myocardial hypertrophy were also measured. In these conditions, compared with controls, rilmenidine reduced blood pressure. This effect was maintained during the treatment period and was associated with a reduction of the hindquarter blood flow resistances. The increase in blood pressure recorded in control rats after 10 days is the last step of the blood pressure increase, which is known to be maximal in 12-week-old SHRs. At the opposite, the initial bradycardia induced by rilmenidine rapidly exhausted. In the present study, we focused on the effects of a lower dose of rilmenidine; we focused on whether, in a particular dose and delivery regimen reducing blood pressure, rilmenidine also reduces the cardiac mass. In our experimental conditions, no significant reduction of the ventricular mass was observed in the rilmenidine-treated rats. The lack of effect of rilmenidine on the left ventricular mass might be a consequence of the too-short duration of treatment or of a low sensitivity of rats toward the antihypertrophic effects or imidazoline-like compounds. Interspecies differences exist in this respect, since a 1-year treatment of patients with rilmenidine reversed left ventricular hypertrophy, 19,20 whereas no antihypertrophic effect could be shown in SHRs 21 and Doca-salt hypertensive rats. 22 Another hypothesis that could explain the lack of effect on myocardial remodeling, could be a too-short-lasting nychthemeral antihypertensive cover.
Although rilmenidine has been shown to be somewhat selective for I1 receptors over α2-adrenergic receptors, this drug reduces blood pressure through a simultaneous action at both α2-adrenergic and I1 receptors. We have previously shown that simultaneous actions on these two types of receptors contribute in a synergic way to reduce blood pressure. 23,24 In rats, this synergism could be blunted because of a low expression of I1 receptors compared with other species such as rabbits. Nevertheless, pharmacokinetic features specific for rats could also explain these observations. In preliminary experiments, we observed that, after a single bolus injection, blood pressure decreased but returned to preinjection values 1.5 to 2 hours after the administration. To study whether this pharmacodynamic profile was linked to a rapid elimination of the compound, plasma titrations were performed in our 2 schedules of administration. Here, we show for the first time that rilmenidine is very rapidly eliminated from the plasma in SHRs. Consequently, the antihypertensive effects of rilmenidine were observed at the peak of the plasma concentration (ie, 30 minutes). Therefore, we can assume that a concentration of nearly 30 ng/ml is required to obtain a blood pressure reduction in SHRs. At this concentration, rilmenidine decreases blood pressure and partially desensitizes the central α2-adrenergic receptors. In the first part of this study, the lack of antihypertensive effect of the infusion of the same total daily dose is probably explained by the too-low plasma concentration. An increase in the daily perfused dose would be possible with the help of minipumps but a continuous delivery of a high dose would probably provoke a progressive escape of the antihypertensive effect due to increased vasoconstriction.
In conclusion, long-term treatment with rilmenidine was able to reduce blood pressure in spontaneously hypertensive conscious and anesthetized rats at least at the peak of the plasma concentration. This hypotensive effect was obtained when the daily dose was divided whereas the continuous delivery was not significantly active, giving a too-low plasma concentration. Therefore, there was no functional desensitization with the b.i.d. procedure. This study shows for the first time the limits of the use of rats for the study of the chronic cardiovascular effects of rilmenidine at the total daily dose of 500 μg/kg. The development of models of cardiac dysfunctions in other species, supposed to be more relevant to humans, is a challenge in this field.
The authors thank the International Research Institute Servier for financial support of this work. We also thank Drs. Michel Dubar and Anne Jacquet (IRIS) for expert assistance.
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