Role of angiotensin II and corticotropin-releasing hormone in hemodynamic responses to cocaine and stress

Regulatory Peptides 127 (2005) 1 – 10 www.elsevier.com/locate/regpep

Review

Role of angiotensin II and corticotropin-releasing hormone in hemodynamic responses to cocaine and stress Mark M. Knuepfer*, Kayla D. Rowe, Julie A. Schwartz, Lance L. Lomax Department of Pharmacological and Physiological Science, St. Louis University School of Medicine, 1402 S. Grand Blvd. St. Louis, MO 63104, United States Received 29 November 2004; accepted 9 December 2004 Available online 11 January 2005

Abstract Cocaine produces characteristic behavioral and autonomic responses due to its unique pharmacological properties. Many of the autonomic responses resemble those to acute behavioral stress. Both cocaine and behavioral stress have been shown to evoke an increase in sympathetic nerve activity that is primarily responsible for the peripheral cardiovascular responses. We noted varying hemodynamic and sympathetic response patterns to cocaine administration and to acute behavioral stress in rats that correlate with the predisposition to develop both a sustained increase in arterial pressure and cardiomyopathies. Several lines of evidence suggest that the autonomic response patterns are dependent on the actions of central peptides including angiotensin II (Ang II) and corticotropin-releasing hormone (CRH). This is based on observations demonstrating that intracerebroventricular (icv) administration of receptor antagonists for Ang II or CRH attenuated the decrease in cardiac output (CO) and increase in vascular resistance noted in some animals after cocaine administration or startle. In contrast, icv Ang II enhances the cardiodepression associated with cocaine administration or startle. Based on this and other evidence, we propose that the autonomic response patterns to startle and to cocaine are closely related and dependent on central Ang II and CRH. Furthermore, we suggest that these central peptides may be responsible for varying predisposition to cardiovascular disease. D 2004 Elsevier B.V. All rights reserved. Keywords: Cocaine; Systemic vascular resistance; Angiotensin; CRH; Cardiovascular responsiveness; Behavioral stress

Contents 1.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Model of predisposition to cocaine-induced cardiovascular toxicity . . . . . . . 2. Corticotropin releasing hormone (CRH) . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Role for CRH in hemodynamic responses to stress and to cocaine. . . . . . . . 2.2. Evidence for the role of CRH in hemodynamic responsiveness to cocaine . . . . 2.3. Evidence for the role of CRH in hemodynamic responsiveness to stress . . . . . 3. Angiotensin II (Ang II). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Role of central angiotensin in hemodynamic responses to cocaine and to stress . 3.2. Evidence for the role of angiotensin in hemodynamic responsiveness to cocaine 3.3. Evidence for the role of angiotensin in hemodynamic responsiveness to startle . 4. Unifying hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

* Corresponding author. Tel.: +1 314 9776462; fax: +1 314 9776411. E-mail address: [email protected] (M.M. Knuepfer). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.12.010

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1. Introduction Cocaine has been used for centuries in South America as a psychostimulant. Within the past 150 years, its use has spread around the world. Early observations of toxic effects led the United States and other countries to limit its use, yet its potent euphorigenic properties have defied curtailment of its abuse by governmental regulation. The effects of cocaine as a local anesthetic and a monoamine reuptake antagonist are well known, yet many untoward responses to cocaine have been described. If there were no toxic responses associated with cocaine use, the studies of its pharmacological effects would have little significance. In fact, it is likely that cocaine is responsible for greater costs to our society than any other illicit drug in the U.S. [1]. In some individuals, cocaine elicits a number of life-threatening cardiovascular responses including myocardial ischemia, arrhythmias, and sudden cardiac death [1,2]. Chronic adverse effects on the myocardium and vasculature have also been noted including enhanced atherosclerosis, cardiac hypertrophy, cardiomyopathies, and apoptosis in cardiac myocytes [3–6]. Until the causes of varying susceptibility to cocaine toxicity in humans are identified, the illicit use of cocaine will continue to assume high costs in both health care and human lives. Cocaine produces a substantial increase in arterial pressure in humans and other animals due to its sympathomimetic actions. Cocaine prolongs the activity of catecholamines by preventing reuptake by the presynaptic nerve terminal [7,8]. Thus, the actions of norepinephrine at the vascular smooth muscle junction are enhanced, resulting in increases in vascular tone and arterial pressure. While the peripheral effects may contribute to the actions of cocaine on the sympathetic nervous system, the central effects appear to be more important in defining the actions of cocaine. Recent studies in humans, dogs, and pigs have shown that direct intracoronary administration of cocaine does not produce significant vasoconstriction, however, systemic or cerebrovascular administration will elicit coronary and systemic vasoconstriction [9–11]. Moreover, it has been shown that the pressor response to cocaine is associated with an increase in sympathetic discharge as noted in humans and animals [12–15]. Therefore, it is likely that many, if not all, of the pharmacological and toxicological effects of cocaine on the cardiovascular system are due to activation of central sympathoexcitatory pathways. Cocaine also elicits behavioral arousal and euphoria in humans and animals [16,17]. These observations suggest that cocaine has potent sympathoexcitatory and behavioral effects due to activation of central sites. Acute behavioral stress or startle elicits characteristic behavioral and autonomic responses that are, in many ways, similar to those evoked by cocaine. In fact, anesthesia attenuates or abrogates many of the hemodynamic and behavioral responses to cocaine [33–36]. Moreover, there

are many similarities in the hemodynamic response patterns to stress and cocaine [18]. The activation of the hypothalamic–pituitary–adrenal (HPA) axis reflected in elevated plasma ACTH and corticosteroids after cocaine administration [19,20] is similar to the responses to stress [21,22]. Sympathetic nerve activity is elevated in response to stress [23] and to cocaine [15]. Likewise, plasma catecholamine levels are increased after cocaine [24,25] and stress [26]. Baroreflex responsiveness is suppressed by cocaine [27,28] and by stress [29]. Chronic stress or cocaine administration elicits increases in dopamine release and turnover in the basal ganglia and these changes exhibit cross-tolerance to one another [30–32]. Therefore, we suggested that the responses associated with use of this psychostimulant were not only dependent on the CNS but were likely to resemble responses to acute stress since both stimuli elicit behavioral arousal requiring a conscious state [37]. These findings implicate some degree of congruence in the central neural pathways mediating autonomic responses to psychostimulants and to behavioral stress. In the past three decades, a number of studies have implicated specific peptides in the CNS that are involved in sympathetic and neurohumoral regulation in response to behavioral stress or psychostimulants. Several experiments have directly demonstrated the contribution of angiotensin II (Ang II) and corticotropin releasing hormone (CRH) to central sympathoexcitatory responses to stress [38–42], but relatively little is known regarding the role of peptides in sympathoexcitatory responses to cocaine. This review article will describe our present understanding of the role of Ang II and CRH in mediating acute hemodynamic and autonomic responses to cocaine administration. We will also compare these hemodynamic responses to those elicited by acute stress or startle. Finally, we will address differences in hemodynamic and autonomic response patterns that we hypothesize result from differences in the central actions of these peptides. Moreover, we propose that the differences in action of central peptides may be responsible for varying predisposition to cocaine- or stress-induced cardiovascular disease. 1.1. Model of predisposition to cocaine-induced cardiovascular toxicity Our laboratory has been studying the CNS causes of hemodynamic response variability to cocaine and to acute stress. We subdivide rats according to their hemodynamic response pattern to cocaine. We define vascular responders as those rats with a pressor response dependent entirely on an increase in systemic vascular resistance (SVR), with a reduction in cardiac output (CO). The remaining rats, designated mixed responders, have an increase in arterial pressure due to a smaller increase in systemic vascular resistance and an increase in cardiac output. Repeated cocaine administration will cause sustained increases in arterial pressure and more severe cardiomyopathies in

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vascular responders compared to mixed responders [37,43,44]. Therefore, we have studied vascular and mixed responders as a model for those individuals that are particularly sensitive to the toxic effects of cocaine [37]. As mentioned earlier, there are many similarities in responses to stress and to cocaine. We reported that an acute startle, elicited by running cold water across the floor of a watertight enclosure, elicits hemodynamic responses in conscious rats that are very similar to the initial responses to intravenous cocaine administration [18]. We observed similar, divergent hemodynamic response patterns in response to startle and to conditioned or unconditioned stressors in conscious rats [18]. This is not surprising since others have described the similarity in responses between stress and behavioral arousal [45]. Therefore, individuals may be identified with specific patterns of hemodynamic and likely neuroendocrine responsiveness to a variety of different perturbations. The significance of hemodynamic response variability to stress was suggested several decades ago. Variability in hemodynamic response patterns to stress has long been recognized in both humans [46–50] and animals [18,51]. It has been reported that humans identified as vascular responders to acute stress are more likely to develop hypertension [46,47,52] and heart disease [53]. In a similar manner, we demonstrated that repeated cocaine administration caused a significant increase in arterial pressure in vascular responders but not mixed responders [37,44]. We also noted more severe cardiomyopathies in response to repeated cocaine administration in vascular responders compared to mixed responders [43]. Moreover, repeated use of a heterologous stress paradigm produced a sustained increase in arterial pressure in vascular but not mixed responders [54]. Therefore, we proposed that the rat may provide a model for studying the causes of varying susceptibility to cardiovascular disease in humans [37]. Using this experimental model, we have sought to understand the causes of hemodynamic response pattern variation. Our data suggest that vascular responders have greater increases in both hindquarters vascular resistance and systemic vascular resistance due to greater increases in sympathetic nerve activity [12,15,37,44,55]. Moreover, ganglionic blockade prevents hemodynamic responses to cocaine [25,56,57]. Therefore, we presumed that the CNS is responsible for different response patterns. Since others suggested that the adverse cardiovascular effects of stress are responsible for the enhanced susceptibility to cardiovascular disease by causing sympathetic hyperactivity [48,49,58–60], we believe that the extent of sympathetic responsiveness to psychostimulants or acute stress may reflect the relative predisposition to cocaine- or stressinduced cardiovascular disease. In order to examine this hypothesis, we investigated the role of central receptors regulating sympathetic outflow and cardiovascular function using intracerebroventricular (icv) injections of selective adrenergic receptor antagonists. We

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reported that yohimbine (3 Ag, icv) attenuates the increase in systemic vascular resistance to cocaine [61]. In contrast, the initial startle response to cold water (1 cm deep) was reduced by prazosin (10 Ag, icv) or phentolamine (30 Ag, icv) but not by yohimbine (3 Ag, icv) pretreatment [62]. Furthermore, propranolol pretreatment (3 or 30 Ag, icv) enhanced the increase in systemic vascular resistance and the decrease in cardiac output in response to cocaine [61] or to startle with cold water [63]. These findings suggest that central a- and h-adrenoceptors mediate hemodynamic responses to cocaine administration and to startle. In this review, we will present recent data implicating a role for CRH and Ang II in mediating variable autonomic and hemodynamic response profiles to cocaine and stress. We will discuss the role of these peptides in defining the unique pattern of hemodynamic responsiveness in rats that covaries with the predisposition to develop cardiovascular disease. These observations implicate both CRH and Ang II as critical peptides in the brain mediating the autonomic response patterns evoked by cocaine or stress in rats.

2. Corticotropin releasing hormone (CRH) 2.1. Role for CRH in hemodynamic responses to stress and to cocaine Corticotropin-releasing hormone (CRH) has been postulated to play an important role in coordinating the endocrine, autonomic, behavioral and immune responses to cocaine or stress through actions in the brain and periphery [38,39,64– 66]. It has long been known that CRH release is an early step in regulating the HPA axis [45,66]. CRH, a 41 amino acid peptide, was isolated and identified as a central peptide that regulates the release of ACTH from the adenohypophysis in response to stress [66,67]. Release of ACTH stimulates corticosteroid production from the adrenal cortex in order to coordinate a systemic humoral response to stress. CRH from neurons located in the parvocellular region of the paraventricular nucleus of the hypothalamus (PVH) are part of the final common pathway for the communication of CNS responses to stress to the pituitary gland. Ablation of the PVH prevents stress-induced ACTH secretion [68]. These changes occur in addition to activation of the sympathoadrenal system to redistribute blood flow, suppress immune function and to cope with a perceived threat or an emotional response. The numerous actions of CRH in stress responsiveness have been reviewed in detail by others [38,39,69,70]. In addition to regulating release of ACTH, central CRH mediates hemodynamic functions. CRH is critical for the autonomic response patterns to acute stress and for baroreflex function [22,38,71]. Intracerebroventricular CRH administration produces an increase in arterial pressure and plasma catecholamines [72,73]. Pretreatment with chlorisondamine, a ganglionic blocker, prevents the

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increase in arterial pressure and heart rate. [74]. Central CRH administration also elicits an increase in sympathetic nerve activity [75,76]. Intracerebroventricular administration of a CRH antibody attenuated the plasma epinephrine response to several different stressors [73,74]. A selective CRH antagonist, a-helical CRF9–41, attenuated the hemodynamic responses to treadmill exercise [77]. Therefore, central CRH not only regulates ACTH and subsequent release of corticosteroids but also mediates sympathoadrenal responsiveness to behavioral stress since icv CRH administration increases sympathetic nerve activity and plasma catecholamines [72,75,76,78]. In a similar manner, cocaine administration produces an increase in ACTH and corticosteroids in animals [20] and in humans [19]. Cocaine stimulates the HPA axis in rat by a CRH-mediated mechanism [65], since cocaineinduced increases in plasma ACTH were inhibited by central injections of CRH antiserum or CRH receptor antagonist [64]. In addition, acute cocaine administration significantly increased CRH mRNA levels, but not arginine vasopressin mRNA levels, in the PVH and amygdala [65]. Cocaine also increases central sympathetic activity by an unknown mechanism [9–11,15]. Therefore, we proposed that central CRH mediates cardiovascular and sympathetic responses to cocaine [61]. These data are described below.

2.2. Evidence for the role of CRH in hemodynamic responsiveness to cocaine Using conscious animals instrumented for arterial pressure and cardiac output determination and for intracerebroventricular drug administration, we examined the role of CRH antagonists on hemodynamic responses to cocaine. We instrumented rats with a miniaturized pulsed Doppler flow probe on the ascending aorta and with femoral arterial and venous cannulas. After recovery from surgery, we characterized rats as vascular or mixed responders according to their hemodynamic response pattern to cocaine (5 mg/kg, i.v., over 45 s) twice daily with a minimum of 3 h between doses. This was repeated for two more days in order to determine whether individual rats were vascular or mixed responders using previously defined characteristics described earlier [37,44]. As reported previously [61], intracerebroventricular administration of the CRH receptor antagonists, a-helical CRF9–41 (10 Ag) or astressin (5 Ag), was capable of attenuating the increase in systemic vascular resistance and the decrease in cardiac output in response to cocaine, particularly in vascular responders. The responses after ahelical CRF9–41 are depicted in Fig. 1. Note that the CRH receptor antagonist selectively attenuated the increase in systemic vascular resistance and the decrease in cardiac

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Fig. 1. The effects of intracerebroventricular administration of vehicle or drugs on the hemodynamic responses to cocaine (5 mg/kg, i.v., infused over 45 s). Data represented are the average of five values (obtained at the time of the peak pressor response and 15, 30, 45, and 60 s after the initial pressor response to cocaine). Both a-helical CRH9–41 (10 Ag, icv, CRHa) or sarthran ([Sar1,Thr8]-angiotensin II, 20 Ag, icv) pretreatment 5 min before cocaine administration attenuated the increase in mean arterial pressure (MAP), systemic vascular resistance (SVR) and the decrease in cardiac output (CO) in vascular responders. In mixed responders, a-helical CRH9–41 or sarthran did not alter these responses. In contrast, angiotensin II (30 ng, icv) administration depressed cardiac output responses to cocaine in all rats and stroke volume responses in mixed responders only. Heart rate responses to cocaine were not affected by these pretreatments (data not shown). The data were examined with a two way analysis of variance and Bonferonni’s post hoc test. Significance was assumed if pb0.05.

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output in vascular responders, while having little effect on mixed responders. Moreover, it prevented the difference in hemodynamic response patterns between vascular and mixed responders despite having little effect on the pressor response to cocaine. These data suggest that CRH may play an important role in mediating varying responsiveness to acute cocaine. It is not yet clear whether preventing the difference in response patterns would alter the development of cardiovascular disease in response to cocaine. 2.3. Evidence for the role of CRH in hemodynamic responsiveness to stress Using a similar approach, we attempted to determine whether central CRH was responsible for the differences in hemodynamic response patterns to acute stress or startle. Rats were instrumented to record arterial pressure and cardiac output as described above. After recovery, rats were tested in watertight enclosures for their responses to brief (1 min) exposure to 1 cm deep ice cold water. The water was added rapidly causing an acute startle and a pressor response not unlike the maximum pressor response to cocaine administration (5 mg/kg, i.v.). Within 15 s, the pressor response was reduced but maintained at a modest increase from baseline for the remainder of the cold water exposure. During this period, heart rate and cardiac output were elevated consistently in all rats. In contrast, the initial response (startle) was characterized by an increase in cardiac output in some rats and a decrease in cardiac output in other

rats, as previously described [18]. We reported previously that hemodynamic response patterns to cocaine in individual rats are closely related to the response patterns elicited by exposure to cold water or to air puff or a conditional stress paradigm [18]. In other words, it appears that vascular responders to acute stress are likely to be vascular responders to cocaine. We also demonstrated that vascular responders to acute stress had a higher arterial pressure after completion of a four week stress exposure compared to mixed responders [54]. We concluded that vascular responders are more likely to develop stress-induced hypertension. Therefore, we studied the causes of differences in hemodynamic responsiveness. As shown in Fig. 2, intracerebroventricular administration of the CRH antagonist a-helical CRF9–41 (10 Ag) prevented the initial decrease in cardiac output in vascular responders with little effect on mixed responders [62]. There was also an attenuation of the increase in systemic vascular resistance after the CRH antagonist (Fig. 2). Therefore, blockade of periventricular CRH receptors was capable of preventing the differences in hemodynamic response patterns to startle much like the effects on responses to cocaine administration. We concluded that central CRH mediates autonomic response patterns to acute stress particularly in vascular responders [62]. It remains to be determined whether these are directly correlated with the predisposition to cardiovascular disease in vascular responders. Currently, we are studying the effects of microinjecting CRH antagonists into specific neuronal sites in the CNS that

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Fig. 2. The effects of intracerebroventricular administration of drugs or vehicle on the initial hemodynamic responses to cold water exposure (startle). Either vehicle (0.9% saline, icv), angiotensin II (30 ng, icv, Ang), losartan (20 Ag, icv, Los) or a-helical CRH9–41 (10 Ag, icv, CRHa) was administered 5 min before addition of ice cold water (1 cm deep) to a watertight plexiglass enclosure (242445 cm high). Those animals with a positive cardiac output (CO) response after saline pretreatment and cocaine were designated mixed responders (n=7) whereas those with a decrease in CO were vascular responders (n=6). Other abbreviations and statistical analyses are described in Fig. 1.

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are known to regulate sympathetic responses to cocaine such as the central nucleus of the amygdala and the dorsomedial hypothalamus. Our preliminary results (n=4 each) suggest that bilateral administration of muscimol (80 pmol) or a-helical CRF9–41 (250 ng) into either area attenuates the decrease in cardiac output in vascular responders (Knuepfer, Bloodgood, and Kucenas, preliminary results). These findings suggest that the decrease in cardiac output unique to vascular responders is dependent on synaptic transmission through the amygdala and dorsomedial hypothalamus and, in part, on CRH receptors in this tissue. These studies will be completed to verify these observations. In summary, there is substantial evidence for a role of CRH in mediating hemodynamic responses to cocaine and to acute stress. More importantly, these data suggest that CRH may be responsible for differences in hemodynamic response profiles that may indicate a susceptibility to cardiovascular disease likely due to sympathetic hyperresponsiveness. Subsequent studies will verify the role of CRH in specific sites in the brain to determine whether central CRH mediates predisposition to heart disease and hypertension.

3. Angiotensin II (Ang II) 3.1. Role of central angiotensin in hemodynamic responses to cocaine and to stress The renin–angiotensin system plays an important role in the regulation and maintenance of arterial pressure and body fluid homeostasis. In the peripheral circulation, it has long been known that Ang II enhances catecholamine release from sympathetic nerve terminals similar to the effects of cocaine [79,80]. This has been proposed to be responsible for enhanced vasoconstriction with circulating Ang II [81]. The direct effects of cocaine administration on plasma renin activity are controversial, since it has been reported by some to decrease [82] and by others to increase plasma renin activity [83]. It has been suggested that converting enzyme inhibition ameliorates toxicity to a lethal dose of cocaine [84]. Therefore, Ang II may be responsible for some of the cardiovascular responses to cocaine. In addition to the peripheral vascular actions of Ang II, it has long been recognized that Ang II is synthesized in the CNS and acts as a neurotransmitter in a number of central projections [85]. The central renin–angiotensin system is known to play a key role in the regulation of autonomic responses and sympathetic responsiveness [40,41]. Central administration of Ang II elicits a pressor response and an increase in sympathetic nerve activity [86–88]. Recently, it was reported that converting enzyme inhibition reduces neuronal vasoconstrictor activity suggesting that CNS angiotensin receptors mediate tonic sympathetic activity [41]. Intravenous Ang II administration increases lumbar

sympathetic nerve activity and arterial pressure in baroreceptor-denervated rats [89]. Therefore, Ang II has a sympathoexcitatory effect that contributes to its pressor response. Angiotensin acts both in the brainstem and in the hypothalamus. Ang II receptors of the AT1 type are located in several brainstem sites known to regulate sympathetic outflow including the rostral ventrolateral medulla, the nucleus tractus solitarius, and the area postrema [90–92]. AT1 receptors are also located in several forebrain sites that regulate arterial pressure and body fluid balance including the paraventricular nucleus, the median preoptic area (mnPOA), the subfornical organ, and the organum vasculosum of the lamina terminalis [90–92]. Ang II acts at several sites including the subfornical organ and the anteroventral third ventricle (AV3V) region [40,93]. Therefore, central angiotensinergic receptors modulate sympathetic outflow and arterial pressure. The central renin–angiotensin system has been implicated as an important component of the autonomic and neuroendocrine responsiveness to behavioral stress [94,95]. Central Ang II receptors are up-regulated by exposure to stress [94,96,97]. Central administration of angiotensin receptor antagonists reduces the cardiovascular and sympathetic responses to stress [98–100]. Losartan administration reduces the stress-induced suppression of baroreceptor function in young rats [71]. These and other studies clearly implicate the central renin–angiotensin system as an important component of the hemodynamic responses to stress. We have argued that several of the autonomic effects of cocaine resemble those to acute stress, particularly startle [18,37]. Therefore, we proposed that central Ang II could play an important role in the central neural integration of hemodynamic responses to cocaine. We have conducted several experiments to verify this hypothesis. 3.2. Evidence for the role of angiotensin in hemodynamic responsiveness to cocaine We compared the effects of intravenous Ang II administration with phenylephrine administration in vascular and mixed responders [101]. While there was not a difference in the baroreflex-mediated bradycardia in response to phenylephrine, mixed responders had significantly reduced reflex reductions in heart rate in response to Ang II administration. Ang II (10–100 ng/kg, i.v.) elicited greater increases in systemic vascular resistance in vascular responders compared to mixed responders. These data suggested that vascular responders may be more sensitive to the vasoconstrictor responses to Ang II. Recently, we sought to determine whether this was dependent on differences in sensitivity of CNS neurons to Ang II. We studied the effects of icv Ang II and an Ang II receptor antagonist on hemodynamic responses to cocaine. After surgical instrumentation and characterization to

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determine whether rats were vascular or mixed responders, rats were treated with icv saline (vehicle) 5 min before administration of cocaine. A minimum of 3 h later, central Ang II or an Ang II antagonist (sarthran or [Sar1,Thr8]angiotensin) were administered intracerebroventricularly 5 min before cocaine administration. Hemodynamic responses, including arterial pressure, heart rate and cardiac output, were compared to responses recorded after intracerebroventricular saline (vehicle). Ang II (30 ng, icv) caused a short lasting pressor response. Subsequent administration of cocaine (5 mg/kg, i.v.) resulted in a greater decrease in cardiac output response in 5 vascular responders with little effect on cardiac output in mixed responders (Fig. 2). In contrast, treatment with the Ang II antagonist, sarthran (20 Ag icv), reduced the decrease in cardiac output and the increase in systemic vascular resistance in 9 vascular responders without affecting 5 mixed responders (Fig. 2). These findings suggest that central Ang II receptors mediate the cardiac output and vascular responses evoked by cocaine in vascular responders but not mixed responders. In fact, the sarthran data suggest that vascular responders may be dependent on central Ang II receptors for greater vasoconstrictor (and presumably sympathetic) responsiveness. Therefore, we propose that cocaine evokes a central sympathoexcitatory response that is dependent on activation of Ang II receptors in the CNS. 3.3. Evidence for the role of angiotensin in hemodynamic responsiveness to startle Using a similar experimental paradigm with exposure to cold water as the stimulus, we examined the effects of intracerebroventricular administration of Ang II or losartan, an AT1 selective receptor antagonist. After characterizing the hemodynamic response pattern to cold water exposure, rats were treated with Ang II (30 ng, icv) 5 min before startle. As expected, Ang II produced a small but significant increase in arterial pressure. Despite this, startle with cold water elicited a pressor response that was similar to vehicle administration (Fig. 2). In contrast, the cardiac output and the stroke volume responses in mixed responders were significantly depressed with little difference in vascular responders (Fig. 2). On a subsequent day, rats were retested several times for their responsiveness to acute cold water exposure before testing them with vehicle, then losartan (20 Ag, icv), preceding startle. Losartan pretreatment had little effect on hemodynamic variables, but subsequent initial responses to cold water demonstrated an attenuation of the increase in systemic vascular resistance (Fig. 2). Moreover, there were no longer differences in the initial arterial pressure, cardiac output, or stroke volume responses to cold water exposure (Fig. 2). These data suggest that Ang II receptor activation in the CNS is, at least in part, responsible for the greater increases in systemic vascular resistance and decreases in

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cardiac output in vascular responders to startle. Therefore, we suggest that activation of central Ang II receptors may be responsible for the greater sympathetic responsivity noted in vascular responders and possibly the greater susceptibility of vascular responders to cardiovascular disease. Ang II receptors in the median preoptic area (mnPOA), a part of the anteroventral third ventricle (AV3V), are critical for the pressor and dipsogenic responses to centrally administered Ang II [102,103]. Moreover, it has been shown that this tissue plays a critical role in the development of a number of different models of experimental hypertension [104]. Others have shown that AT1, but not AT2 receptors, are expressed in this tissue [105]. Therefore, we examined the possibility that administration of an AT1selective antagonist into the mnPOA would alter hemodynamic responses to cocaine or to startle [106]. Rats were instrumented with a guide cannula over the median preoptic area, a flow probe on the ascending aorta, and arterial and venous cannulas. After recovery, we administered losartan (20 ng in 100 nl over 1 min) into the mnPOA 10 min before cocaine administration (5 mg/kg, i.v.) in 8 vascular responders. The decrease in cardiac output and increase in systemic vascular resistance were significantly reduced by losartan, suggesting that hemodynamic responses were dependent on activation of AT1 receptors in this tissue (data not shown). Likewise, administration of losartan (20 ng in 100 nl over 1 min) significantly attenuated the increase in systemic vascular resistance and decrease in cardiac output in 6 vascular responders in response to startle (cold water). These data suggest that the sympathoexcitation in response to cocaine or to startle is dependent on activation of AT1 receptors in the mnPOA. Since vascular responders are more prone to develop stress- or cocaine-induced cardiovascular disease [37,43,44], we propose that this tissue plays an important role in mediating this predisposition.

4. Unifying hypothesis Our data support the contention that CRH and Ang II play important roles in determining the hemodynamic responses to cocaine and to startle. A number of investigators have reported compelling evidence that the release of CRH is regulated by Ang II [67,100,107,108]. In conscious rats, Ang II administration evokes a dose-related increase in plasma ACTH that is abolished by immunoneutralization with CRH antibody [107]. In addition, the presence of mRNA for AT1 receptors is found exclusively in neurons with mRNA for CRH [108]. Others have reported that Ang II is not required for the stress response since central administration of Ang II inhibitors did not prevent the rise in ACTH and corticosterone in response to ether stress [109]. Despite this, the majority of evidence suggests a critical interaction between the two peptide neurotransmitters. More importantly, our preliminary evi-

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dence suggests that central Ang II may mediate the differences in hemodynamic responsiveness between vascular and mixed responders. We have proposed that the subset identified as vascular responders has greater sympathetic activation in response to cocaine [15]. Central Ang II evokes sympathoexcitation [89] and plays a key role in the sympathetic responses to acute stress [41,96,110]. Therefore, we propose that a neural pathway dependent on Ang II and CRH neurotransmission is activated by cocaine or startle and is responsible for the hemodynamic response variability noted in individual rats. If the central sympathomimetic effects of cocaine are dependent on CRH and Ang II receptors in the CNS, it is likely that these peptides are also responsible for the predisposition of some individuals towards the development of cardiovascular disease. We reported that vascular responders are prone to cocaine-induced cardiomyopathies and to cocaine- or stress-induced hypertension [37,43,44,54]. Future studies will determine whether these central peptides are responsible for individual susceptibility to develop cardiovascular disease.

Acknowledgments The authors would like to thank Dr. Qi Gan for assistance with these experiments. Some of these data were previously published [61] or published in abstract form [106,111]. We gratefully acknowledge the generous contribution of losartan from Merck Research Laboratories. This work was supported by USPHS grants DA05180 and DA13256 and by the American Heart Association, Heartland Affiliate.

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