Effect of co-administration of morphine and nicotine on cardiovascular function in two-kidney one clip hypertensive (2K1C) rats

Abstract

Cardiovascular morbidity and mortality are potentiated with smoking and hypertension. The aim of this study was to investigate the effects of morphine and nicotine co-administration on cardiovascular function in two-kidney one-clip hypertensive (2K1C) rats. Thirty-two male rats were divided into four groups as follow: Vehicle, morphine, nicotine and nicotine + morphine. All drugs were administered for 8 weeks. Baroreflex sensitivity (BRS), heart rate and blood pressure were measured using a Power Lab data acquisition. Plasma rennin activity (PRA) and serum concentration of nitric oxide (NO) were measured using Elisa method. To induce hypertension, the renal artery of left kidney was clipped for 8 weeks. A significant decrease in systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) was observed in nicotine + morphine group compared to vehicle and nicotine groups (p<0.05). Serum concentration of NO was lower in nicotine + morphine group compared to morphine group and significantly higher than nicotine group. The BRS was lower in the nicotine + morphine group compared to other groups. The PRA level was higher in nicotine + morphine compared to morphine group but it was higher than nicotine group. This study demonstrated that prolonged co-consumption of morphine and nicotinedecreased PRA and blood pressure and increased the serum concentration of NO in hypertensive rats. Co-administration of morphine and nicotine decreased BRS in 2kic hypertensive rats probably via central nervous system.

INTRODUCTION

Today smoking behavior is among it the most accepted preventable causes of death in the United States. Smokers experience a wide range of physiologic side effects that increase the risk of cardiovascular disease (CVD) [1]. It is widely accepted that hypertension and smoking are of potent independent risk factors considered for cardiovascular morbidity and mortality, and concomitant smoking in hypertensive patients may increase the risk of coronary heart disease [2, 3]. Prolonged cigarette smoking increased BP, systemic vascular resistance and heart rat [4]. Nitric oxide (NO) is an inevitably important molecule in control of vascular tone, blood flow, peripheral vascular resistance, and systemic blood pressure. Chronic smoking impairs endothelial function via lowering the formation of NO [5]. It is proposed that dysfunction of NO pathway in hypertensive subjects is a consequence rather than a cause of elevated BP [6]. Recent evidences obtained from human based studies showed that smoking of four cigarettes per hour impairs baroreflex sensitivity [7]. Opioid receptors demonstrated to regulate cardiovascular function in both normal and diseased myocardium [8]. It has been reported that systemic administration of morphine decreases MAP in the rat and evokes orthostatic hypotension in the healthy patient [9]. Moreover, morphineinduces NO formation in the endothelium and some other tissues [10]. Recent investigations showed the importance of NO during baroreflex function development in hypertensive rats [11]. Several studies examined the effect of morphine and/or nicotine on cardiovascular system while no studies was undertaken on the effect of co-administration of morphine and nicotine on cardiovascular system. Therefore, in this study we examined the effect of co-administration of morphine and nicotine in the development and/ or maintenance hypertension and BRS in hypertensive rats.

MATERIAL AND METHODS

In the present study thirty two male Wistar rats weighing 200±20 grams were enrolled. Rats were accommodated at a controlled temperature with free access to food and water. Prior to start of treatment, animals were randomly divided into below 4 different groups: (1): vehicle group (received only saline), (2): morphine group (received 3 mg/ kg of morphine), (3): nicotine group (received 0.01 mg/ kg of nicotine) and (4): nicotine combined with morphine (received 3 mg/kg of morphine and 0.01 mg/kg of nicotine). All of drugs were administered for a period of 8 weeks.

Induction of hypertension in rats by 2K1C goldblatt method

Initially rats were anesthetized using intraperitonealy ketamine hydrochloride (60 mg/kg) and xylazine (7.5 mg/kg). The left kidney was exposed via flank incision and a silver clip with internal gap of 0.2 mm was put around the renal artery. The sham group, were also treated by this procedure except using silver clip. To control the risk of infections, rats received penicillin G (25000 IU/rat) after surgery. Systolic and diastolic blood pressure was measured once a week by a tail-cuff plethysmography under light ether anesthesia. After 8 weeks of treatment, the animals were anesthetized and direct blood pressure was measured by a catheter (PE50) inserted into femoral artery. Blood samples were collected to measure plasma rennin activity (PRA) in further analysis.

Measurement of Plasma renin activity

The PRA was measured by a kit using 125 I Angiotensin-I generation. Angiotensin Icoated-tube radioimmunoassay (RIA) wasperformed in two aliquots of the same sample, one incubated at 37 °C for generation and onenon-incubated; PRA was calculated as ngAngiotensin I generated/ml/h (Renctk P2721, Sorin-Biomedica Diagnostic Division RIA kit, Italy). The PRA assay sensitivity was 0.13 ng/ml; intra-and interassay coefficients ofvariations were 7.5 and 7.7%, respectively.

Measurement of serum NO

Following 8 weeks of induction of hypertension, blood samples were collected from all animals. The serum NO concentration was measured by Gris reagent system (Promega Corporation, Madison, USA). Serum sample, were added to the wells (96-well flat-button enzymatic assay-plate). A sulfanilamide solution was then added to the wells pre-treated with sample and following of additionN-1-naph-tylethylenediamine dihydrochloride (NED) to the wells under acidic conditions. Finally, the absorbance were read in 520-550 nm by a microreader [12]. The NO concentration in the samples was determined and compared to NO standard curve. The limit of detection was 2.5 μM nitrite.

Baroreflex Assessment

The direct AP and HR were recorded in anesthetized rats. Catheters were connected to a pressure transducer (Maxxim Medical, Athens, TX) coupled to a multichannel recorder by a custom-designed amplifier (University of Iowa, Iowa City, IA). The analog input was converted into a digital signal using a Power Lab data acquisition system (AD Instruments, Mountain View, CA). The MAP was derived electronically using a low-pass filter set at 100 Hz and was calculated using the cyclic mean. Data were acquired at 200 samples/s. HR was determined by measuring the number of heartbeats triggered from the arterial pressure pulse and was calculated online. Once the animal had sufficient time to adapt to the surrounding environment of in the testing chamber, hemodynamic parameters were monitored for 20-40 min to ensure stabilization of MAP and HR. Subsequent to the confirmation of stable hemodynamic parameters, the baseline parameters were continuously recorded for at least 10 min. The baroreflex was tested with a pressor dose of phenylephrine (PE bolus: 8 μg/kg i.v.; Sigma Chemical) and a depressor dose of sodium nitroprusside (SNP bolus: 50 μg/kg i.v.; Sigma Chemical). The baroreflex was calculated as the ratio of HR variations to MAP variations (ΔHR/ΔMAP). There was an interval of at least 15 minutes between the infusions to allow the recovery of basal values [13].

Statistical analysis

The results are presented as Mean±S.E.M. Data were analyzed using ANOVA followed by the tukey post-hoc test. Differences were considered statistically significant if p value was less than 0.05.

RESULTS

BP and HR

The SBP, DBP and MAP were decreased significantly in morphine combined + nicotine group compared to vehicle and nicotine groups (p<0.01). Administration of morphine was also significantly decreased DBP, MAP and SBP compared to vehicle group. HR was not significantly differed among groups (Figure 1 and 2).

FIGURE 1.

Variations in SBP, DBP and MAP in experimental groups.

*Significant difference in SBP and DBP between morphine+ nicotine with vehicle and nicotine group (all p<0.05)

# Significant difference in SBP and DBP between morphine with vehicle and nicotine group (all p<0.05). SBP; systolic blood pressure, DBP; systolic blood pressure, MAP; mean arterial pressure. Data are shown as mean ± S.E.M

FIGURE 2.

Variation in HR in experimental groups.

PRA measures

The PRA was significantly higher in morphine + nicotine group compared to morphine treated animals (p<0.01). PRA in morphine group was decreased compared to vehicle group (p<0.001) while, in nicotine group it was higher than vehicle group (Figure 3).

FIGURE 3.

Variations of PRA in experimental groups.

*Significant difference between morphine with all other three groups (p<0.05).

# Significant difference between morphine+ nicotine with morphine and nicotine groups (all p<0.05).

Serum NO concentration

The serum concentration of NO was increased in morphine treated animals compared to vehicle. In morphine + nicotine group the serum level of NO was also higher than vehicle group (p<0.05) however, in nicotine treated animals it decreased compared to vehicle group (p<0.05) (Figure 4).

FIGURE 4.

Variations in serum level of NO in experimental groups.

*Significant difference between morphine with all other three groups (p<0.05).

# Significant difference between morphine+ nicotine with morphine and nicotine groups (p<0.05).

Effects of morphine and nicotine on BRS

In morphine treated group the BRS was increased compared to vehicle group (p<0.05). Treatment with nicotine has no significant effect on BRS (compared to vehicle) while, this index was significantly decreased in morphine + nicotine group compare to all groups (all p<0.05) (Figure 5).

FIGURE 5.

Variations in BRS in experimental groups.

**Significant difference between morphine+ nicotine with all other three groups (all p<0.01).

DISCUSSION

Present findings demonstrated that prolonged co-administration of nicotine and morphine decreased PRA and blood pressure in hypertensive rats while administration of nicotine increased PRA and blood pressure in these animals. In addition, in these animals, the serum concentrations of NO were increased following chronic consumption of morphine alone or in combination with nicotine. Accumulating evidences indicated that in early phase of 2K1C hypertensive animals both increased activity of PRA and renin-angiotensin-aldosterone and sympathetic activity are responsible for increasing blood pressure [14, 15, 16]. In addition, subsequent to eight weeks of clipping, altered endothelial function is also of notable factors in regulation of hypertension. Nicotine has established to enhance the Ang-II receptor mediated vasotrophic effect [17, 18], BP response to angiotensin II (Ang-II) and increase Ang II–induced contractions of aortas and mesenteric arteries [18]. Our results also demonstrated that application of nicotine increased BP and PRA in 2K1C hypertensive animals. NO is certainly one of the most important endothelium originated factors. NO is a derivative of L-arginine which is converted in L-citrulline and NO. NO has multiple beneficial functions in vascular homeostasis including vascular tone and modulation of BP. Endothelial dysfunction, which is described as decreased NO bioavailability, is considered among important risk factors for hypertension. Abnormalities in endothelium-derived relaxing factors especially NO and impaired endothelium-dependent relaxation in human clinical and several animal models of hypertension have been well demonstrated [19-21]. A recent study indicated that morphine stimulated secretion of NO and anti-hypertensive role has been reported for endogenous morphine-NO signaling events [22]. Therefore, morphine possibly modulates endothelial function and vascular en-dothelial cells function and influences the local paracrine-autocrine regulatory pathway by endogenously expressed authentic morphine, thus, causes constitutive NO expression. Moreover, considerable evidences revealed that the NO signalling plays paramount role in opioid receptor-mediated responses in the neurocardiovascular system [23, 24]. It has been well documented that the smoking behavior is of the main risk factors for coronary vascular diseases including, hypertension. Chronic smoking impairs endothelial function by inhibiting the formation of NO in parallel with increasing the degradation of NO via generation of free oxygen radicals [5]. Furthermore, nicotine down-regulates eNOS, an enzyme involved in the pathway of NO generation, decreases endothelium dependent vasodilatation, and in turn stimulates the adhesion of leukocytes to the endothelium to induce VED and further atherosclerosis [25-27]. In addition, nicotine induces hypertension by stimulating the release of catechol-amine [28]. Our results demonstrate that following concomitant administration of morphine and nicotine the serum NO concentration was more than the group that received only nicotine. So, one can conclude that morphine may inhibited the effects of nicotine on NO in hypertensive rats.

In current set of experiments we also examined the effect of co-administration of morphine and nicotine on BRS and observed a decreased in BRS in hypertensive rats. Some studies indicated that endogenous and exogenous opioids regulate the performance of BRS through effects on central opioids receptors [9]. In habitual smokers, besides vasopathic [29] and myopathic processes, alterations in the function of the autonomic blood pressure control should be considered as possible cause of cardiovascular diseases [30]. Abnormalities of the vascular barorecptors as well as glossopharyngeal (the vagal) nerve can be responsible for the loss of BRS [31]. During these pathologic circumstances, the nervous BP control can be abolished so that extreme fluctuation of ABP may occur. Indeed, recently smoking-induced alterations of the autonomic nervous system, especially a disturbance of the sympatho-vagal balance, were reported [32]. During this two suggested probable mechanisms for this sympatho-vagal imbalance: Chronic nicotine (tobacco) abuse leads to a restricted sensitivity of the afferent baroreceptors or directly restricts the baroreflex-centers in the brain stem [7]. While a diminished perfusion of the brain leading orthostatic hypotension, an elevated sympathetic tone corresponds to reduce BRS [32], the limited compensation of the smoke induced catecholamine-peaks causes a labile hypertension [31]. However, we expected morphine stimulates release of NO from endothelial cell, increase arterial compliance, hence, decreases BP and consequently improves BRS. A recent study indicated smoking increases the heart rate, systolic, mean and diastolic blood pressure, and decreases the baroreflex sensitivity [33]. Cigarette smoking increases the activity of the autonomic nervous system (the sympathetic part), increases heart rate and blood pressure and impairs the baroreflex [34, 35]. The mechanism of increasing blood pressure and heart rate by nicotine is believed to be by activation of the sympathetic nervous system [36] or by effects on endothelial function [37]. Nicotine diminishes the baroreflex gain probably via reducing arterial compliance and stretch receptor responsiveness [38]. The direct interaction of nicotine with central mechanisms integrating the baroreceptor input into autonomic responses is another plausible mechanism for the depressant action of nicotine on baroreflexes [39]. A third mechanisms the ability of nicotine to modify the effectors responsiveness to reflex autonomic modulation [30]. We expect that increase arterial compliance was due to increase NO level in the combination of morphine and nicotine leading increase BRS, But we observed that BRS decrease in this group. Thus, it is possible that the decrease BRS witch was observed in the combination of morphine and nicotine was due to direct interaction of morphine and nicotine whit central mechanisms integrating the baroreflex function.

CONCLUSION

In conclusion, this study demonstrated that prolonged combined treatment with morphine and nicotine decrease PRA and BP while increase the serum concentration of NO in hypertensive rats. Also administration of morphine prevented induction of hypertension probably via NO pathways. Coadministration of morphine and nicotine decreased BRS in 2k1c hypertensive rats probably via central nervous system.

DECLARATION OF INTEREST

The authors declare no conflict of interest for present study.