Alamandine injected into the paraventricular nucleus increases blood pressure and sympathetic activation in spontaneously hypertensive rats

Abstract

The intricate and multifaceted renin-angiotensin system (RAS) plays a fundamental role in regulating diverse physiological processes, most notably cardiovascular homeostasis and fluid balance. Within this complex hormonal cascade, alamandine has recently emerged as a newly discovered and compelling component, demonstrating distinct biological activities. Preliminary investigations have indicated that alamandine possesses vasoactive properties in specific regions of the nervous system, hinting at its broader neuromodulatory potential. The primary objective of the present study was to thoroughly investigate whether the precise administration of alamandine directly into the hypothalamic paraventricular nucleus (PVN), a crucial brain region involved in cardiovascular control, modulates systemic blood pressure and efferent sympathetic nervous system activity.

To achieve this, experiments were meticulously conducted on anesthetized rats, where continuous measurements of mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) were precisely recorded. The investigation included both normotensive Wistar-Kyoto (WKY) rats, serving as a control group, and spontaneously hypertensive rats (SHRs), which represent a well-established genetic model of essential hypertension. Our findings revealed that the microinjection of alamandine into the PVN elicited a significant and robust increase in both MAP and RSNA in both WKY rats and SHRs. Notably, the magnitude of these cardiovascular and sympathetic responses was considerably more pronounced and exaggerated in the SHRs, suggesting a heightened sensitivity or altered regulatory mechanism in the hypertensive state.

To delineate the underlying molecular mechanisms mediating these effects, further experiments employed specific pharmacological antagonists. Crucially, the observed increases in MAP and RSNA induced by alamandine were entirely blocked or substantially attenuated by pretreatment with D-Pro7-Ang-(1-7), a recognized antagonist of the alamandine receptor Mas-related G-protein-coupled receptor, member D (MrgD). Furthermore, the sympathoexcitatory and pressor effects were also abrogated by the prior administration of SQ22536, a potent inhibitor of adenylyl cyclase (AC), and by rp-adenosine-3′,5′-cyclic monophosphorothionate (Rp-cAMP), a specific inhibitor of protein kinase A (PKA). These results strongly implicate the MrgD receptor and the subsequent activation of the cyclic AMP (cAMP)-PKA signaling pathway in alamandine’s action within the PVN.

Intriguingly, when D-Pro7-Ang-(1-7), SQ22536, or Rp-cAMP were administered unilaterally into the PVN of SHRs in isolation, these agents consistently resulted in a significant reduction in both mean arterial pressure and renal sympathetic nerve activity from their elevated basal levels. This compelling observation suggests that the MrgD/cAMP-PKA pathway might exhibit constitutive overactivity in the PVN of SHRs, thereby contributing to their elevated sympathetic tone and hypertension. Conversely, direct microinjection of exogenous cAMP alone into the PVN of these animals caused a notable increase in both MAP and RSNA, further confirming the positive role of this signaling molecule in cardiovascular regulation. Moreover, pretreatment with cAMP prior to alamandine administration synergistically enhanced alamandine’s pressor and sympathoexcitatory effects, providing definitive evidence for the involvement of the cAMP-PKA pathway as a downstream mediator. Taken together, these comprehensive results unequivocally indicate that the targeted microinjection of alamandine into the hypothalamic paraventricular nucleus increases systemic blood pressure and augments sympathetic outflow primarily through the activation of the MrgD receptor and its subsequent signaling via the cAMP-PKA pathway. These findings offer novel insights into the neurohumoral regulation of blood pressure and sympathetic tone, particularly in the context of hypertension.

Keywords: Alamandine; Hypertension; Hypothalamic paraventricular nucleus; Sympathetic activation; cAMP-PKA pathway.

Introduction

The intricate neural and humoral systems that govern physiological homeostasis are often perturbed in various pathological states, leading to significant alterations in autonomic nervous system activity. A substantial body of evidence consistently demonstrates a heightened sympathetic drive in numerous clinical disorders, including but not limited to patients suffering from essential hypertension, a complex multifactorial condition without a clear single cause. This sympathetic hyperactivity is also prominently observed in cases of obesity-related secondary hypertension, where metabolic dysfunction directly contributes to elevated blood pressure, and in individuals afflicted with chronic kidney disease, a condition that frequently manifests with increased sympathetic tone. Furthermore, this phenomenon is not confined to human pathologies; it is recapitulated in well-established experimental models of hypertension, such as spontaneously hypertensive rats (SHRs), which serve as a genetic paradigm for human essential hypertension, and in two-kidney one-clip hypertensive rats, a model of renovascular hypertension. The enduring and excessive sympathetic activation seen across these diverse conditions is a critical factor contributing to the initiation and perpetuation of hypertension, as well as the progressive development of associated end-organ damage, highlighting its central role in disease pathogenesis.

A key brain region with paramount importance in the central regulation of blood pressure and sympathetic activity is the hypothalamic paraventricular nucleus (PVN). This highly integrated neuroanatomical structure serves as a crucial autonomic control center, orchestrating cardiovascular responses through its extensive and direct projections. These efferent pathways extend notably to the rostral ventrolateral medulla (RVLM), a region recognized as a primary source of sympathetic vasomotor tone, and also descend to the intermediolateral column of the spinal cord, where sympathetic preganglionic neurons originate. Importantly, the PVN has been definitively implicated in mediating the chronic, excessive sympathetic activation characteristic of sustained hypertensive states. Its persistent overactivity actively participates in driving the pathogenesis of hypertension and exacerbating the relentless progression of organ damage that commonly accompanies chronically elevated blood pressure, affecting vital organs such as the heart, kidneys, and blood vessels.

The renin-angiotensin system (RAS) stands as a highly complex and multifaceted endocrine system that plays an unequivocally vital role in the meticulous regulation of the cardiovascular system and the maintenance of electrolyte and fluid balance throughout the body. In recent years, our understanding of the RAS has expanded significantly beyond its classical components, with the discovery and characterization of several novel elements. These exciting additions to the RAS family include the prorenin/renin receptor, which mediates the tissue-specific activation of renin; angiotensin-converting enzyme-2 (ACE2), an enzyme that metabolizes angiotensin II to angiotensin-(1-7); and the angiotensin-(1-7)/Mas receptor axis, which is generally associated with counter-regulatory, protective cardiovascular effects. Among these more recently characterized components, the vasoactive peptide alamandine has been identified as a new member within the broader RAS family. Structurally, alamandine exhibits a striking similarity to angiotensin-(1-7), differing by only a single amino acid substitution at its amino terminus, where an alanine residue replaces an aspartate. This subtle yet significant structural difference imbues alamandine with its distinct biological properties. Alamandine can be generated through two primary enzymatic pathways: either through the decarboxylation of angiotensin-(1-7) or, alternatively, through the hydrolysis of angiotensin A by ACE2, underscoring its intricate integration into the expanded RAS enzymatic network.

Previous investigations into alamandine’s effects within the central nervous system have yielded intriguing, regionally specific results. It has been demonstrated that the microinjection of alamandine into the rostral ventrolateral medulla (RVLM), a key sympathoexcitatory brainstem region, produces a pressor effect, leading to an increase in blood pressure. Conversely, when microinjected into the caudal ventrolateral medulla (CVLM), a region primarily involved in sympathetic inhibition, alamandine elicits a depressor effect, causing a reduction in blood pressure. Furthermore, these observed effects, whether pressor or depressor, have been unequivocally shown to be mediated by the specific receptor for alamandine, which has been identified as the Mas-related G-protein-coupled receptor, member D (MrgD). Crucially, these effects can be specifically blocked by the administration of the antagonist D-Pro7-Ang-(1-7), confirming the involvement of the MrgD receptor. However, despite these advancements, the precise role that alamandine plays within the hypothalamic PVN remains entirely unknown. Specifically, it has not yet been elucidated whether alamandine exerts any influence over the regulation of systemic blood pressure and sympathetic activity within the PVN, particularly in the context of hypertension, a major gap in our understanding of its cardiovascular actions.

Recent compelling studies have shed light on the intracellular signaling pathways potentially involved in RAS component actions within central cardiovascular control regions. For instance, a notable study demonstrated that angiotensin-(1-7) in the RVLM enhances the cardiac sympathetic afferent reflex, ultimately leading to an increase in sympathetic output and elevated blood pressure, a process mediated via the cyclic AMP (cAMP)-protein kinase A (PKA) pathway. Similarly, another independent study suggested that alamandine itself possesses the capacity to reverse hyperhomocysteinemia-induced vascular dysfunction, a beneficial effect that also appears to be mediated through the PKA signaling pathway. Given these established roles of the cAMP-PKA pathway in cardiovascular regulation and alamandine’s known actions, it is plausible that if alamandine indeed plays a significant role in the pathophysiology of hypertension within the PVN, then the cAMP-PKA signaling cascade might be intimately involved in its mechanism of action. Accordingly, the overarching aim of the present study was precisely to determine whether alamandine exerts an effect within the PVN to increase blood pressure and sympathetic activation in spontaneously hypertensive rats (SHRs). Furthermore, a critical secondary objective was to ascertain, if such an effect were observed, whether the well-established cAMP-PKA intracellular signaling pathway is mechanistically involved in mediating these physiological responses.

Materials And Methods

Animals

All experimental procedures were meticulously carried out using 12-week-old male rats, specifically obtained from Vital River Biological Co., Ltd in Beijing, China. For the purposes of these experiments, two distinct strains of rats were utilized: normotensive Wistar-Kyoto (WKY) rats, which served as a crucial control group representing a non-hypertensive state, and spontaneously hypertensive rats (SHRs), which constitute a widely recognized and robust genetic model of essential hypertension, allowing for direct comparison of responses in normotensive versus hypertensive conditions. All animal handling, experimental protocols, and surgical procedures were subjected to rigorous scrutiny and received explicit approval from the Experimental Animal Care and Use Committee of Nanjing Medical University. Furthermore, every effort was made to ensure full compliance with the ethical guidelines and stringent standards outlined in the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised 1996), underscoring a commitment to animal welfare and responsible research conduct. The rats were housed in a carefully controlled environment, maintaining a constant ambient temperature and operating on a precise 12-hour light-dark cycle to regulate their circadian rhythms. Throughout the experimental period, all animals had unrestricted and free access to standard laboratory chow and tap water, ensuring their nutritional and hydration needs were consistently met.

Acute Experiment

For the acute experimental phase, the rats were initially anesthetized through the intraperitoneal administration of a combination of urethane (at a dose of 800 mg/kg) and α-chloralose (at a dose of 40 mg/kg). This specific anesthetic regimen was chosen for its ability to provide stable, long-lasting anesthesia with minimal impact on cardiovascular and sympathetic nervous system functions, which were the primary parameters under investigation. Throughout the duration of each experiment, supplemental doses of these anesthetic agents, typically one-tenth of the initial dose, were administered intravenously as needed. The necessity for these supplemental doses was continuously monitored and assessed by evaluating the animal’s depth of anesthesia, primarily indicated by the absence of corneal reflexes and a lack of the paw withdrawal response to a noxious pinch stimulus. This ensured a consistent and adequate level of surgical anesthesia, minimizing discomfort. Following the induction of anesthesia, rats were intubated and mechanically ventilated with room air using a specialized rodent ventilator to maintain stable respiration and oxygenation throughout the procedure. For the continuous and precise measurement of mean arterial pressure (MAP), the right carotid artery was surgically cannulated. This cannula was then directly connected to a high-fidelity pressure transducer, which in turn was integrated into a data acquisition system. This setup allowed for the accurate, real-time, and continuous recording of systemic blood pressure throughout the experimental period, providing critical cardiovascular data.

Recording Of Renal Sympathetic Nerve Activity

To obtain direct and quantifiable measurements of sympathetic nervous system outflow, the renal sympathetic nerve activity (RSNA) was meticulously recorded. This procedure commenced with a careful retroperitoneal incision to expose the left renal sympathetic nerve. Following its successful isolation, the renal nerve was surgically sectioned distally, towards the kidney. This distal cut was a critical step designed to eliminate any potential confounding afferent (sensory) activity originating from the kidney, ensuring that the recorded signals solely represented efferent sympathetic outflow from the central nervous system. The isolated nerve was then carefully placed onto a pair of specialized silver electrodes, which were meticulously positioned to optimize signal capture. To ensure stable electrical recording and to prevent desiccation, the nerve and electrodes were then fully immersed in warm mineral oil. The raw RSNA signals captured by the electrodes were then fed into an AC/DC differential amplifier, specifically designed for neurophysiological recordings. The amplifier was configured with a low-frequency cutoff set at 10 Hz and a high-frequency cutoff at 3000 Hz, allowing for optimal signal-to-noise ratio for sympathetic nerve activity. Both the raw, unfiltered electrical activity and the integrated RSNA signal were simultaneously recorded onto a PowerLab data acquisition system, providing comprehensive data capture. The amplified and filtered signals were subjected to electronic integration with a time constant of 1.0 second, providing a smoothed, quantifiable representation of overall nerve activity. At the conclusion of each experiment, the baseline background noise was precisely determined by sectioning the central end of the nerve, thereby eliminating any physiological sympathetic signals. This background noise value was then consistently subtracted from all integrated RSNA values, ensuring the accurate quantification of true sympathetic nerve activity and enhancing the reliability of the measurements.

PVN Microinjections

For the precise delivery of pharmacological agents to the hypothalamic paraventricular nucleus (PVN), the rats were securely positioned in a stereotaxic frame, a specialized apparatus that allows for accurate and reproducible targeting of specific brain regions. The stereotaxic coordinates for the PVN were meticulously determined based on a standard rat brain atlas, specifically Paxinos & Watson’s, and were established as 1.8 mm caudal to bregma, 0.4 mm lateral to the midline, and 7.9 mm ventral to the dorsal surface of the skull. The volume of microinjection was rigorously controlled at 50 nanoliters for each side of the bilaterally targeted PVN. To minimize any potential time-dependent physiological changes or diffusion, bilateral PVN microinjections were completed swiftly, within a timeframe of 1 minute. At the conclusion of each experiment, to ensure the accuracy of the microinjection site, the identical volume of 2% Evans Blue dye was injected into the presumed microinjection location. This allowed for subsequent microscopic histological identification of the injection site within the brain tissue. Data from any rats where the microinjection sites were found to be outside the predetermined boundaries of the PVN were stringently excluded from the final data analysis. Additionally, animals were also excluded from analysis if the measured distance between the precise center point of the microinjection and the nearest boundary of the PVN was found to be less than 0.15 mm, ensuring that only data from accurately targeted injections were considered, thus maintaining the integrity and reliability of the experimental results.

Chemicals

All chemical compounds utilized in this study were acquired from reputable commercial suppliers to ensure high purity and consistent quality. Alamandine, the novel vasoactive peptide under investigation, was obtained from Phoenix Pharmaceuticals located in California, USA. D-Pro7-Ang-(1-7), which functions as a specific antagonist for the alamandine receptor MrgD, was procured from Bachem Chemical Company, also situated in California, USA. Angiotensin II (Ang II), along with Rp-adenosine-3′,5′-cyclic monophosphorothionate (Rp-cAMP), a well-characterized inhibitor of protein kinase A (PKA), cyclic AMP (cAMP) itself, and 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536), a potent inhibitor of adenylyl cyclase (AC), were all obtained from Sigma, located in Missouri, USA. All these essential chemicals were carefully dissolved in normal saline solution, ensuring their physiological compatibility for microinjection into the brain tissue and maintaining consistent osmotic conditions.

Experimental Protocols

Rats used in this study were systematically and randomly assigned into a total of 13 distinct experimental groups, with each group comprising 6 animals (n = 6), ensuring adequate statistical power and minimizing bias. Microinjections into the PVN were performed according to a predefined set of experimental protocols to administer various pharmacological agents. These included control injections of normal saline, three different doses of alamandine (specifically 4 picomoles, 40 picomoles, and 400 picomoles) to assess dose-dependency, a single dose of angiotensin II (40 picomoles) for comparative purposes, D-Pro7-Ang-(1-7) (50 picomoles), cAMP (1 nanomole), SQ22536 (2 nanomoles), and Rp-cAMP (1 nanomole). Furthermore, complex experimental conditions involved the pretreatment of the PVN with D-Pro7-Ang-(1-7), cAMP, SQ22536, or Rp-cAMP, followed by the administration of alamandine. Specifically, alamandine was administered 5 minutes after pretreatment with D-Pro7-Ang-(1-7), and 8 minutes after pretreatment with cAMP, SQ22536, or Rp-cAMP. The precise doses for all compounds used in this study were carefully selected based on efficacy and established concentrations reported in previous relevant research, ensuring their biological relevance and effectiveness within the experimental model.

Statistical Analyses

All experimental data obtained from this study are consistently presented as the mean value plus or minus the standard error of the mean (SEM), providing a clear indication of central tendency and variability within the datasets. Statistical analyses were meticulously performed using GraphPad Prism 4.0 software, a widely recognized and robust statistical package developed by GraphPad Software Inc., located in California, USA. The primary statistical tests employed were either one-way or two-way ANOVA (Analysis of Variance), depending on the complexity of the experimental design and the number of independent variables. When multiple comparisons were made across different experimental groups, the ANOVA was consistently followed by a Bonferroni post hoc test. This specific post hoc analysis was chosen to adjust for multiple comparisons, thereby reducing the likelihood of Type I errors and enhancing the stringency of statistical inference. A two-tailed P-value of less than 0.05 was prospectively established as the threshold for considering statistical significance, indicating that observed differences were unlikely to have occurred by chance.

Results

Microinjection Of Alamandine Increases The MAP And RSNA, And These Effects Are Blocked By An Alamandine Receptor Antagonist

Our initial investigations focused on characterizing the dose-dependent effects of alamandine when microinjected directly into the hypothalamic paraventricular nucleus (PVN). We administered three distinct doses of alamandine (4, 40, and 400 pmol) and observed a clear dose-dependent increase in both mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). Notably, both the middle (40 pmol) and high (400 pmol) doses of alamandine elicited statistically significant increases in MAP and RSNA in both normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). A crucial observation was that the magnitude of these effects, both on MAP and RSNA, was consistently greater in SHRs compared to WKY rats, suggesting a heightened sensitivity or an exaggerated response to alamandine in the hypertensive state. For comparative purposes, angiotensin II (Ang II) was also microinjected into the PVN, and it similarly increased MAP and RSNA in both WKY rats and SHRs. Consistent with the alamandine findings, these effects of Ang II were also more pronounced in SHRs than in WKY rats. Interestingly, there were no statistically significant differences observed between the pressor and sympathoexcitatory responses induced by alamandine and those induced by Ang II, indicating comparable potency in these effects. Furthermore, it was observed that alamandine microinjected into the PVN had no significant effect on heart rate in either WKY rats or SHRs, suggesting a specific modulation of blood pressure and sympathetic outflow without direct chronotropic effects.

To further elucidate the specific receptor mediating these observed effects of alamandine, we next administered D-Pro7-Ang-(1-7), a recognized antagonist of the alamandine receptor MrgD, into the PVN via microinjection. We made a significant finding that D-Pro7-Ang-(1-7) alone caused a measurable decrease in both MAP and RSNA specifically in SHRs, but it exerted no statistically significant effect on either parameter in WKY rats. This strongly suggests a basal overactivity of the MrgD receptor pathway contributing to hypertension in SHRs. Moreover, and critically, pretreatment of the PVN with D-Pro7-Ang-(1-7) completely blocked or substantially attenuated the sympathoexcitatory and pressor effects of alamandine on both MAP and RSNA in both WKY rats and SHRs. These results provide compelling evidence that the actions of alamandine in the PVN are indeed mediated through its specific receptor, MrgD.

The cAMP-PKA Pathway Mediates The Effects Of Alamandine On MAP And RSNA

To thoroughly investigate the direct involvement of the cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway in mediating the observed cardiovascular and sympathetic effects of alamandine, we proceeded with microinjections of exogenous cAMP directly into the PVN. To this end, we consistently observed a notable increase in both the mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) in both normotensive WKY rats and spontaneously hypertensive rats (SHRs). Significantly, the increases induced by cAMP were demonstrably greater in SHRs compared to WKY rats, suggesting an enhanced responsiveness of this pathway in the hypertensive state. Moreover, a crucial finding was that pretreatment of the PVN with exogenous cAMP prior to alamandine administration synergistically enhanced the effects of alamandine on both MAP and RSNA in both WKY rats and SHRs, providing strong evidence for the cAMP-PKA pathway operating as a downstream mediator or amplifier of alamandine’s actions.

Conversely, to further explore the role of this pathway, microinjections of SQ22536, a specific inhibitor of adenylyl cyclase (AC), the enzyme responsible for cAMP synthesis, were performed. This intervention resulted in a significant decrease in both RSNA and MAP specifically in SHRs, but it had no statistically significant effect on either parameter in the WKY rats. This observation provides further support for the concept of basal overactivity within the cAMP pathway contributing to the elevated sympathetic tone and hypertension in SHRs. Furthermore, pretreatment with SQ22536 in the PVN entirely abolished the alamandine-mediated increases in MAP and RSNA in both WKY rats and SHRs, providing strong evidence that AC activity and subsequent cAMP production are essential for alamandine’s effects.

Finally, to specifically pinpoint the involvement of PKA, the downstream effector of cAMP, we performed microinjections of Rp-cAMP, a known PKA inhibitor, directly into the PVN. We consistently found that Rp-cAMP effectively attenuated both MAP and RSNA in SHRs, whereas it exerted no statistically significant effect on either parameter in the WKY rats. This further supports the role of PKA overactivity in sympathetic dysregulation in SHRs. Importantly, pretreatment with Rp-cAMP in the PVN significantly inhibited the effects of alamandine on both MAP and RSNA in both WKY rats and SHRs. These results collectively and robustly demonstrate that the pressor and sympathoexcitatory actions of alamandine within the PVN are mechanistically mediated through the activation of the cAMP-PKA signaling pathway.

Discussion

Previous foundational studies have consistently identified the hypothalamic paraventricular nucleus (PVN) as a critical and highly integrated vasomotor center within the central nervous system. This vital brain region plays an indispensable role in the meticulous control of arterial pressure and the regulation of basal sympathetic nerve activity, exerting a profound influence over cardiovascular homeostasis. Furthermore, extensive research has previously reported that the direct microinjection of various members of the renin-angiotensin system (RAS) family, such as angiotensin II (Ang II) and angiotensin-(1-7) (Ang-(1-7)), into the PVN leads to measurable increases in systemic blood pressure and heightened sympathetic nerve activity. However, alamandine, a peptide that has only recently been discovered and characterized as a novel component within the expanding RAS, has remained largely uninvestigated regarding its specific functional role within the PVN. The present study was meticulously designed to address this knowledge gap, and its compelling findings definitively demonstrate that the precise microinjection of alamandine into the PVN significantly increases systemic blood pressure and markedly enhances sympathetic activity. Crucially, these observed effects are shown to be mediated via its specific receptor, the Mas-related G-protein-coupled receptor, member D (MrgD), and are further confirmed to involve the intracellular cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway as a key mediator.

Alamandine has previously been the subject of research exploring its diverse biological actions. Prior investigations have established that it exerts significant vasoactive effects in distinct regions of the nervous system, demonstrating its capacity to influence blood vessel tone centrally. Furthermore, studies involving its oral administration in hypertensive animal models have revealed its ability to induce long-term antihypertensive effects, underscoring its therapeutic potential. These beneficial effects have been consistently shown to be mediated by the MrgD receptor and are effectively abrogated by its specific antagonist, D-Pro7-Ang-(1-7). In the current study, consistent with these broader systemic actions, the microinjection of alamandine directly into the PVN resulted in a notable increase in both mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) in both normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). A particularly significant finding was the heightened sensitivity observed in SHRs, where the increases in MAP and RSNA were considerably more pronounced compared to those in WKY rats, suggesting that the alamandine-MrgD pathway may be dysregulated or hypersensitive in the hypertensive state. Moreover, to rigorously confirm the receptor specificity of alamandine’s actions, the microinjection of D-Pro7-Ang-(1-7), the MrgD receptor antagonist, into the PVN yielded multifaceted results. It not only attenuated both MAP and RSNA when administered alone in SHRs, implying a constitutive activation or heightened tone of this pathway in hypertension, but it also effectively and comprehensively blocked the alamandine-induced increases in MAP and RSNA in both WKY rats and SHRs. This collective body of evidence definitively demonstrates that the administration of exogenous alamandine into the PVN increases systemic blood pressure and renal sympathetic nerve activity, that these effects are unequivocally mediated by the MrgD receptor, and that alamandine exhibits a greater potency in spontaneously hypertensive rats compared to their normotensive WKY counterparts.

Building upon the identification of the MrgD receptor as the mediator of alamandine’s effects, our subsequent phase of investigation aimed to delineate the downstream intracellular signaling cascades involved, specifically focusing on whether the cAMP-PKA pathway participates in alamandine’s actions. To achieve this, we employed highly selective pharmacological tools: SQ22536, a potent and membrane-permeable inhibitor of adenylyl cyclase (AC), the enzyme responsible for the synthesis of cAMP, which effectively and completely blocks the increase in cAMP levels evoked by AC activation; and Rp-cAMP, a specific and membrane-permeable inhibitor of protein kinase A (PKA), which directly antagonizes the effects of cAMP by preventing PKA activation. In the present study, the direct microinjection of exogenous cAMP alone into the PVN resulted in measurable increases in both MAP and RSNA in both WKY rats and SHRs. Notably, this effect was significantly more pronounced in SHRs, suggesting that the cAMP pathway itself may contribute more substantially to sympathetic activation and blood pressure regulation in the hypertensive context. Furthermore, a crucial observation that strengthens the link between alamandine and the cAMP-PKA pathway was that pretreatment of the PVN with exogenous cAMP markedly enhanced the pressor and sympathoexcitatory effects of alamandine on MAP and RSNA in both rat models. This enhancement was, once again, demonstrably greater in SHRs, providing compelling evidence that the cAMP-PKA pathway acts as a crucial downstream signaling mechanism or an amplifier for alamandine’s actions within the PVN, particularly in the context of hypertension.

Intriguingly, the inverse experiment, involving the pretreatment of the PVN with either SQ22536 (the AC inhibitor) or Rp-cAMP (the PKA inhibitor), completely abolished the alamandine-induced increases in MAP and RSNA in both WKY rats and SHRs. This complete blockade provides robust and unequivocal evidence that the cAMP-PKA pathway is an essential and indispensable mediator of alamandine’s sympathoexcitatory and pressor effects within the PVN. Moreover, a particularly insightful finding emerged when SQ22536 or Rp-cAMP were microinjected alone into the PVN: both agents significantly attenuated the basal levels of MAP and RSNA specifically in SHRs, yet they had no statistically significant effect on these parameters in normotensive WKY rats. This differential response strongly suggests that the cAMP-PKA pathway exhibits a constitutive overactivity or elevated basal tone in the PVN of SHRs, and this heightened activity critically contributes to the maintenance of high blood pressure and sympathetic activation characteristic of spontaneous hypertension. Our findings are in strong agreement with previous independent studies that have demonstrated that PKA inhibitors can significantly curtail the ability of alamandine to exert its vasoactive effects in other physiological contexts, further reinforcing the universal involvement of this signaling cascade.

In conclusion, the findings of this study provide novel and significant insights into the central neurohumoral regulation of blood pressure and sympathetic outflow. We unequivocally demonstrate that the administration of alamandine into the hypothalamic paraventricular nucleus leads to a robust increase in systemic blood pressure and a heightened sympathetic output, primarily through the activation of its specific receptor, MrgD. Furthermore, a key observation is that alamandine exerts more pronounced and exaggerated effects in spontaneously hypertensive rats compared to normotensive WKY rats, suggesting a heightened sensitivity or an altered set point in the hypertensive state. This increased responsiveness is likely linked to the finding that MrgD activity within the PVN appears to be greater in SHRs than in WKY rats. Finally, our study provides definitive evidence that the cyclic AMP-PKA signaling pathway plays a crucial mediating role in alamandine’s effects within the PVN. This direct involvement strongly suggests that this particular intracellular pathway contributes significantly to the elevated blood pressure and chronic sympathetic activation observed in spontaneous hypertension, thereby opening new avenues for understanding and potentially targeting the underlying mechanisms of this prevalent cardiovascular disease.