Biphasic effects of single-dose intravenous injection of uridine adenosine tetraphosphate on blood pressure in mice

Abstract

Purpose

To explore the effects of a single dose of uridine adenosine tetraphosphate (Up₄A) administered through the tail vein, on the blood pressure of mice.

Methods

The mice were separated into three groups: the Up₄A group, the norepinephrine (NA) group, and the α, β-methylene adenosine triphosphate (α, β-meATP) group. Each group of mice were injected drugs through the tail vein at 1, 3, 10, and 30 nmol/kg doses in an ascending order. Additionally, six mice were injected Up₄A through the tail vein at 20, 40, 60, and 80 nmol/kg doses in an ascending order. The administration intervals for each dose were 20 min.

Results

Mice in these groups experienced a rapid increase in blood pressure, reaching its peak within 10 s after drug administration. It took approximately 120 s for the blood pressure to return to baseline levels after the administration of the drugs in both the NA and α, β-meATP groups. After higher doses of Up₄A were administered to the mice, their blood pressure exhibited biphasic changes. Initially, blood pressure of the mice rapidly dropped to a minimum within 10 s, then rose rapidly to a peak within 30 s. Subsequently, it gradually declined, taking around 10 min to return to the levels before the drug administration.

Conclusion

Compared to NA and α, β-meATP, Up₄A, which contains purine and pyrimidine components, displayed a weaker blood pressure-elevating potency. Through its corresponding structure, Up₄A exerted vasodilatory and vasoconstrictive effects throughout the entire experiment resulting in biphasic changes in blood pressure.

Introduction

According to the Global Burden of Diseases study, hypertension (HTN) and its subsequent complications are responsible for 12% of the overall disability-adjusted life years (DALYs) and constitute 63% of the DALYs attributed to cardiovascular disorders in China [1]. Elevated systolic blood pressure remains a principal determinant for premature mortality among patients diagnosed with cardiovascular diseases. In the year 2021, elevated systolic blood pressure was directly implicated in the demise of approximately 10.8 million patients diagnosed with cardiovascular diseases (95% CI 9.15–12.1 million) and was associated with the mortality of 11.3 million patients from somatic causes (95% CI 9.59–12.7 million). Given the profound implications of hypertension for global health, the pursuit of specific and effective control measures and treatments is both an imperative and challenging endeavor [2].

Uridine adenosine tetraphosphate (Up₄A) was first identified by Jankowski in 2005. Jankowski recognized its origin from endothelial cells, categorizing it as a vasoconstrictor. It was postulated that Up₄A mediates its vasoconstrictive biological effects predominantly through the activation of P2X₁ receptors present in vascular smooth muscles [3]. Subsequent research by a team headed by Jankowski in 2007 delineated notably elevated plasma concentrations of Up₄A in hypertensive adolescents compared to their normotensive counterparts, with mean concentrations being 33 nmol/L and 4 nmol/L, respectively [4]. Furthermore, there was a significant correlation between Up₄A levels, left ventricular mass, and the medial thickness of the arterial intima. Intriguingly, Up₄A is distinguished as the first dinucleotide encompassing both purine and pyrimidine configurations. Subsequent research has consistently indicated its pivotal role in modulating vascular tone [3]. Investigations spearheaded by Tólle postulate that among purinergic vasoactive mediators, Up₄A stands out as the most efficacious [5]. Its primary mechanism of action centers on the induction of vasoconstriction via P2X₁ receptor activation. Furthermore, research by Matsumoto et al. ascertained that Up₄A elicits pronounced contractions in several arteries, including the basilar, renal, and femoral arteries, particularly in deoxycorticosterone acetate (DOCA)-salt rats, an established experimental model for salt-dependent arterial hypertension [6, 7]. Concurrently, evidence suggests an upregulated expression of angiotensin type 1 receptor (AT1R) and P2X₁ receptor proteins in angiotensin II (AngII)-induced hypertensive murine models [8].

The purine component of Up4A is postulated to play a role in the pathogenesis of hypertension, possibly mediated through specific receptor interactions. Drawing upon observations from Hansen et al., it was determined that an exposure to 10 μmol/L of Up₄A elicits a contraction of 18 ± 6% in the smooth muscle cells of the murine thoracic aorta previously pre-contracted with phenylephrine [9]. This is subsequently followed by a relaxation phase of approximately −46 ± 6%. Notably, concentrations of Up₄A below 10 μmol/L appeared inert, neither inducing contraction nor relaxation in this vascular segment. This has led to the hypothesis that the dual vasodilatory and vasoconstrictive actions of Up₄A may be attributed to its purine and pyrimidine structures respectively, each potentially acting on its cognate receptors.

Norepinephrine (NA) functions as an α1 receptor agonist and is frequently utilized in clinical settings as a vasoactive agent to elevate blood pressure in patients. Within the vascular endothelial cells, the P2Y receptors predominantly facilitate vasodilation, whereas the P2X₁ receptors located in the vascular smooth muscle are the principal purinergic receptors responsible for mediating vasoconstriction [10]. Adenosine triphosphate (ATP) acts as the agonist for the P2X₁ receptor. However, in studies focusing on vasoconstriction mediated by the P2X₁ receptor, α, β-meATP emerges as a pivotal pharmacological instrument. Chronic exposure to α, β-meATP can result in P2X₁ receptor desensitization [11]. In light of this, our current investigation employed a singular dose administration methodology to assess the impacts of NA, α, β-meATP, and Up₄A on blood pressure within the carotid arteries of anesthetized mice via caudal vein injection. The overarching objective was to elucidate and corroborate the role of Up₄A in modulating vascular tone and the initiation of hypertension.

Material and methods

Up4A was procured from Biolog Life Science Institute, Germany. Both α, β-meATP and NA were sourced from Sigma Company, United States. The employed pressure transducer (MLT0380/D) alongside the eight-channel physiological recorder (Powerlab/8sp) were products of the AD Instrument Company, Australia. All chemicals, including NA, α, β-meATP, and Up₄A, were prepared as 0.1 mol/L solutions and subsequently stored under refrigeration. Kunming strain mice, adhering to the clean grade and weighing between 35 to 42 g, were furnished by the Experimental Animal Center situated in Hebei Province. 2. The study was approved by the Institutional Review Board of the Ethics Committee of Affiliated Hospital of Hebei University. All animals were treated in compliance with the National Research Council’s Guide for the Care and Use of Laboratory Animals (1996).

Part 1: Effects of different doses of α, β-meATP, NA, and Up4A on the blood pressure in the carotid arteries of mice

In this investigation, a total of eighteen Kunming strain mice of clean grade were used. They were categorically divided into three distinct groups, with each group consisting of six mice: the α, β-meATP group, the NA group, and the Up₄A group. Anesthesia was administered to these mice using urethane at a dosage of 1.5 g/kg via intraperitoneal injection. Subsequent to this, their tail veins were accessed and tracheal intubation was performed to ensure airway patency. Their right common carotid arteries were meticulously isolated and cannulated. Once arterial cannulation was successfully achieved, a stabilization period of 20 min was allowed. A pressure transducer was then linked to the experimental mice, and fluctuations in blood pressure within their common carotid arteries, both pre and post drug administration, were captured using an eight-channel physiological recorder. In a systematic ascending protocol, each mouse across the three groups was administered their respective drugs in dosages of 1, 3, 10, and 30 nmol/kg, ensuring a 20 min interlude between consecutive doses [12].

Changes in systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean blood pressure (MBP) (mm Hg) within the common carotid arteries, both pre and post drug administration, were meticulously recorded. For the determination of these blood pressure values, the following methodology was employed: The minimum values for each parameter during the 2 min intervals both before and after the administration of the drug were used to represent the respective blood pressure metrics. Data were continuously accumulated over a 2 s period, from which an average value was subsequently computed.

Part 2: Effects of Up₄A at different doses on the blood pressure of the common carotid arteries of mice.

Based on the observations from the initial phase of the experiment, it was evident that only the 10 and 30 nmol/kg dosages of Up₄A led to modest elevations in the blood pressure of mice. Consequently, the dosage gradient for the Up₄A group was revised to encompass 20, 40, 60, and 80 nmol/kg. In this revised protocol, each mouse was subjected to an intravenous injection of Up₄A in a progressive dosage sequence—20, 40, 60, and 80 nmol/kg. A consistent 20-min gap was maintained between the administration of each successive dose. The influence of each Up₄A dosage on the mice blood pressure was closely monitored, capturing variations in specific blood pressure parameters of the common carotid arteries both pre and post drug administration. The subsequent steps in the experimental process remained unchanged from the original protocol.

Statistical processing

The blood pressure measurements for the mice are presented as the mean ± standard deviation ($\overline{x }$± s). A two-way analysis of variance (ANOVA) was employed to scrutinize the impact of varying drugs on blood pressure. Comparisons between different drug interventions at an identical dosage were facilitated by a one-way ANOVA. In instances where P < 0.05, a subsequent analysis was undertaken using the Dunnett’s test. All statistical analyses, as well as graphical renderings, were conducted using the GraphPad Prism 5.0 software.

Results

Part 1: The effects of NA, α, β-meATP, and Up4A on the blood pressure of common carotid arteries of mice.

Upon intravenous administration through the tail vein of both NA and α, β-meATP at concentrations of 1, 3, 10, and 30 nmol/kg, there was a dose-dependent augmentation in the SBP, DBP, and MBP of the common carotid artery. Subsequent to the drug introduction, an acute surge in blood pressure was observed, peaking within a span of 10 s. This heightened pressure swiftly subsided, culminating in a complete reversion to baseline levels by 120 s (as depicted in Fig. 1a, b). Conversely, the intravenous injection of Up₄A at dosages of 1 and 3 nmol/kg demonstrated inconsequential impacts on the blood pressure measurements within the common carotid arteries of the mice (illustrated in Fig. 2). However, the administration of Up₄A at elevated concentrations of 10 and 30 nmol/kg manifested a modest elevation in the said blood pressure metrics (also illustrated in Fig. 2).

Fig. 1

Changes in the blood pressure in the common carotid arteries of mice that were intravenously injected with NA (30 nmol/kg, a), α, β-meATP (30 nmol/kg, b), and Up₄A (80 nmol/kg, c)

Fig. 2

The dose-dependent effects of NA, α, β-meATP, and Up₄A on the increase in the SBP of the common carotid arteries in mice (**P < 0.01, n = 6)

When subjected to intravenous injection at dosages of 1, 3, 10, and 30 nmol/kg, a dose-dependent elevation in mouse SBP was observed for all three drugs. Statistical analysis revealed marked differences in the blood pressure response across the three drug groups (P < 0.01, as represented in Fig. 2). Hierarchically, the potency of the rise in SBP was as follows: α, β-meATP > NA > Up₄A (**P < 0.01, n = 6, Fig. 2). Specifically, at a concentration of 30 nmol/kg, the respective increases in SBP values facilitated by α, β-meATP, NA, and Up₄A were 64.14 ± 16.28 mmHg, 36.71 ± 10.26 mmHg, and 6.10 ± 2.13 mmHg, respectively. Notably, the disparities in the blood pressure augmentation between Up₄A and α, β-meATP, and between Up₄A and NA were both statistically significant.

Upon administering the drugs intravenously at concentrations of 1, 3, 10, and 30 nmol/kg, a pronounced dose-dependent surge in mouse DBP was evident for all the three therapeutic agents. The intergroup differences in the DBP response were statistically significant (P < 0.01, as depicted in Fig. 3). In terms of the magnitude of DBP elevation, the order was determined to be: α, β-meATP > NA > Up₄A (** P < 0.01, n = 6, Fig. 3). Specifically, when administered at a dosage of 30 nmol/kg, α, β-meATP, NA, and Up4A augmented DBP values by 63.86 ± 12.16 mmHg, 32.86 ± 11.04 mmHg, and 8.70 ± 3.37 mmHg, respectively. There were significant discrepancies in the DBP augmentations elicited by Up₄A compared to both α, β-meATP and NA.

Fig. 3

The effects of intravenous injections of α, β-meATP, NA, and Up₄A at doses of 1, 3, 10, and 30 nmol/kg via on the increase in DBP in the common carotid arteries in mice (^**P < 0.01, n = 6)

Following the intravenous administration of the drugs at concentrations of 1, 3, 10, and 30 nmol/kg, a clear dose-dependent escalation in mouse MBP was observed for all three drugs. This elevation exhibited statistically significant variances across the three groups (P < 0.01, n = 6, Fig. 4). Hierarchically, the magnitude of MBP elevation followed the sequence: α, β-meATP > NA > Up₄A (** P < 0.01, n = 6, Fig. 4). When introduced at a dosage of 30 nmol/kg, the increases in MBP values induced by α, β-meATP, NA, and Up₄A were 63.95 ± 13.19 mmHg, 34.14 ± 10.57 mmHg, and 7.84 ± 2.91 mmHg, respectively. The differences in in the induced increase in blood pressure between Up4A and both α, β-meATP and NA proved to be statistically significant.

Fig. 4

The dose-dependent effects of NA, α, β-meATP, and Up₄A on the increase in the MBP in the common carotid arteries of mice (**P < 0.01, n = 6)

Upon intravenous administration of NA, α, β-meATP, and Up4A at a standardized dosage of 30 nmol/kg, the relative potency in elevating various metrics of mouse blood pressure was observed to be: α, β-meATP > NA > Up₄A (** P < 0.01, n = 6, Fig. 5). Specifically, the increments in MBP facilitated by α, β-meATP, NA, and Up4A at this dosage were 63.95 ± 13.19 mmHg, 34.14 ± 10.57 mmHg, and 7.84 ± 2.91 mmHg, respectively. Statistical analysis revealed significant differences amongst the three drug groups. (When compared to Up4A, the difference was significant at ** P < 0.01; and compared to NA, the significance level was ## P < 0.01 n = 6, Fig. 5).

Fig. 5

The effects brought about by the administration of α, β-meATP, NA, and Up₄A at a dose of 30 nmol/kg through intravenous injection on the increases in SBP, DBP, and MBP in the common carotid arteries in mice (Compared to Up₄A, **P < 0.01. Compared to NA, ^##P < 0.01, n = 6)

Part 2: The effects of different concentrations of Up4A on the SBP, DBP, and MBP within the common carotid arteries of the mice.

Following the administration of Up₄A, a marked decline in blood pressure was observed, reaching its nadir within a 10 s timeframe. This decline in blood pressure was promptly succeeded by a surge, peaking approximately at the 30 s mark. Thereafter, the blood pressure began its gradual reduction, ultimately reverting to baseline values around 10 min post-injection (as depicted in Fig. 1c). The response to varying dosages of Up₄A (20, 40, 60, and 80 nmol/kg) administered intravenously via the tail vein demonstrated a clear dose-dependent effect, both in the initial decrease in blood pressure and subsequent increase. This was evident both in the initial decrement of blood pressure and its subsequent amplification. Notably, the magnitude of the blood pressure post-Up4A administration was less pronounced than the ensuing rise in pressure. Moreover, the influence of Up₄A was more discernible on the diastolic pressure as compared to the systolic pressure (n = 6, Fig. 6).

Fig. 6

Effects of various doses of Up₄A on lowering and elevating SBP, DBP, and MBP values in the common carotid arteries of mice (n = 6)

When 20 nmol/kg of Up₄A was intravenously injected, the reductions in SBP, DBP, and MBP were 5.6 ± 2.3, 4.6 ± 1.14, and 4.93 ± 1.44 mmHg, respectively; the increases were 3.20 ± 1.64, 5.4 ± 3.65, and 4.67 ± 2.92 mmHg, respectively (n = 6). When 40 nmol/kg of Up₄A was intravenously injected, the reductions in SBP, DBP, and MBP were 8.6 ± 2.3, 10.8 ± 3.35, and 10.07 ± 2.98 mmHg, respectively; the increases were 9.00 ± 3.16, 12.00 ± 3.08, and 11.00 ± 2.89 mmHg, respectively (n = 6). When 60 nmol/kg of Up₄A was intravenously injected, the reductions in SBP, DBP, and MBP were 10.4 ± 1.82, 13.6 ± 4.83, and 12.53 ± 3.47 mmHg, respectively; the increases were 15.00 ± 4.00, 20.00 ± 4.18, and 18.33 ± 4.00 mmHg, respectively (n = 6). When 80 nmol/kg of Up₄A was intravenously injected, the reductions in SBP, DBP, and MBP were 11.8 ± 2.59, 17.60 ± 3.44, and 15.67 ± 2.66 mmHg, respectively; the increases were 21.60 ± 8.08, 26.60 ± 9.04, and 24.93 ± 8.34 mmHg, respectively (n = 6, Fig. 7).

Fig. 7

The effect of Up₄A at a dose of 80 nmol/kg on the blood pressure in the common carotid arteries of mice (*P < 0.05, **P < 0.01, n = 6)

Discussion

Although significant progress has been made in understanding the natural course of primary hypertension over the past several decades, and risk factors for hypertension have been identified by epidemiological studies, the specific pathogenesis remains an active area of research and has not yet been fully elucidated [13,14,15]. Among the recognized factors that impact arterial blood pressure are the following: (1) stroke volume; (2) heart rate; (3) peripheral resistance; (4) the elastic reservoir function of the aorta and large arteries; and (5) blood volume. Any change in any of these factors may result in a change in blood pressure. An increase in peripheral resistance causes an increase in the amount of blood held in the aorta at the end of diastole of the left ventricle, resulting in a considerable increase in diastolic pressure, assuming all other parameters remain constant. Therefore, the increase in diastolic pressure generated by the contraction of vascular smooth muscle is more pronounced than the increase in systolic pressure.

In the current investigation, it was observed that the hypertensive responses elicited by α, β-meATP, an agonist of the P2X1 receptor, and NA, an agonist of the α1 receptor, were markedly superior to the effects induced by Up₄A. At lower dosages of Up₄A (1, 3, 10, and 30 nmol/kg), there was an absence of significant changes in the blood pressure within the carotid arteries of anesthetized mice. However, upon a revised dosing regimen of 20, 40, 60, and 80 nmol/kg, notable hemodynamic responses were observed. Specifically, post-administration of Up₄A, there was a swift decrement in blood pressure, hitting its nadir within 10 s. This was subsequently followed by a rapid ascent, culminating in a peak at about the 30 s mark, before initiating a steady decline, ultimately reverting to baseline around 10 min post-injection. Both the precipitous decline and the ensuing escalation in blood pressure exhibited clear dose-responsiveness. The magnitude of the initial blood pressure decline was comparatively subdued compared the subsequent rise.

An intriguing observation from this study was the more pronounced effect of Up₄A on diastolic pressure as compared to systolic pressure. Specifically, at an intravenous dose of 80 nmol/kg of Up₄A, the recorded reductions in blood pressure in the mice were 11.8 ± 2.59 mmHg for SBP, 17.60 ± 3.44 mmHg for DBP, and 15.67 ± 2.66 mmHg for MBP. These reductions were then succeeded by increases of 21.60 ± 8.08 mmHg, 26.60 ± 9.04 mmHg, and 24.93 ± 8.34 mmHg in the same respective parameters. The more potent influence of Up₄A on diastolic pressure may be attributed to its enhanced constriction of peripheral blood vessels, thereby elevating peripheral vascular resistance. This supposition is in alignment with the known physiological relationship between vascular resistance and diastolic pressure, whereby a heightened resistance typically leads to a more pronounced increase in diastolic pressure. The biphasic vascular effects of Up₄A, encompassing an initial contraction followed by relaxation and then reconstruction at elevated concentrations, have previously been identified by research groups led by Hansen and Tolle. Specifically, in the experiment conducted by Hansen, upon administering a concentration of 10–5 mmol/L of Up₄A cumulatively to mice, a biphasic response was observed in their dissected thoracic aortas that had been pre-contracted [9]. This consisted of an initial, transient contraction (19 ± 6%), followed by a more substantial relaxation (46 ± 6%). However, as the Up₄A concentration was increased to 5 × 10–5 mmol/L, a renewed vasoconstrictive response emerged.

Tolle et al. used a model involving healthy Wistar-Kyoto rats with kidneys perfused with vascular angiotensin AngII in vitro [16]. Their findings indicated that continuous Up₄A infusion induced a biphasic vascular response characterized by an initial constriction followed by a subsequent relaxation. Both the constriction and relaxation phases were dose-dependent. The constriction phase elicited by Up₄A was attenuated by the application of non-selective P2R antagonists (namely suramin and PPADS) and a selective P2X₁ receptor agonist (α, β-meATP). This suggests that the P2X₁ receptor plays a pivotal role in mediating the constrictive effect of Up₄A. Conversely, the relaxation phase prompted by Up₄A was counteracted by non-selective P2R antagonists (specifically suramin, PPADS, and RB2) and a selective P2Y₁R antagonist (MRS2179). These results indicate that the P2Y₁R or P2Y₂R is central in mediating the vasodilatory effect of Up₄A on renal vessels. Interestingly, the biphasic effect of Up₄A was also discerned in our experiment. However, there were clear distinctions from previous studies. In our case, Up₄A initiated a decline in blood pressure, followed by a swift ascent. This discrepancy may be attributed to divergent experimental designs. While Hansen and Tolle conducted investigations on isolated tissues (specifically the thoracic aorta of mice and the kidneys of rats in vitro perfused with vascular angiotensin AngII), our study employed an in vivo approach. Drawing upon the research by Jankowski, it has been demonstrated that compared to an equivalent dose of NA, Up₄A prompts a more sustained elevation in blood pressure, a finding that aligns well with our experimental outcomes [5].

Interestingly, the administration of Up₄A demonstrated varied hemodynamic responses based on the experimental setup and conditions. In an experiment involving a single dose of 100 nmol/kg of Up₄A, administered directly into the thoracic aortas of anesthetized rats, only a consistent elevation in arterial pressure was observed without any corresponding drop in blood pressure. This contrasts the effects documented by Hansen et al., wherein a continuous infusion of Up₄A at a rate of 512 nmol/min/kg via the femoral vein of awake mice resulted in a substantial reduction of femoral artery pressure, plummeting from an average of 99 ± 4.0 mmHg down to 64 ± 7 mmHg [9]. Remarkably, this decrease was not accompanied by any subsequent elevation in blood pressure. Therefore, in the aforementioned studies, a monophasic blood pressure response—either a singular increase or decrease—was the dominant observation. This stands in stark contrast to the biphasic response (both an increase and a subsequent decrease or vice versa) noted in our study. The discrepancies in the hemodynamic outcomes across these experiments may be attributed to the different methodologies employed, particularly the routes and methods of Up₄A administration. Each of these techniques could influence vascular tone differently, leading to the distinct observed effects. The complexities of the vascular system, coupled with the intricacies of the physiological responses to Up₄A, emphasize the need for nuanced and contextual interpretations when comparing results across different experimental conditions.

In other experiments related to isolated arterial vessels and organs, Jankowski, et al. administered the P2X₁ receptor agonist—α, β-meATP to anesthetized rats, which significantly inhibited the vascular constriction induced by Up₄A [4]. This suggests that the P2X₁ receptor is involved in the constrictive effect of Up₄A on perfused isolated rat kidneys. Jankowski et al. used Up₄A isolated from epithelial cells of renal tubules in both humans and pigs to perfuse the isolated kidneys of C57BL/6 mice [17]. They discovered that the specific P2X₁ receptor inhibitor (inosine monophosphate, Ip5I) antagonized the constrictive effect of Up₄A on the renal afferent arterioles. This suggests that in the isolated afferent arterioles of C57BL/6 healthy mice, the constrictive effect of Up₄A is mediated by the P2X₁ receptor. The study by Linde et al. revealed that cumulative administration of Up4A ((10^–9–10^–5 mmol/L) brought about relaxation (approximately 25%) in the aorta cycle of SD rats whose endothelia were intact and blood vessels were preliminarily constricted using phenylephrine [18]. In the control group of rats whose endothelia were denuded, Up₄A revealed no significant relaxation effect. This indicates that the vasodilatory effect of Up₄A on the aortas of SD rats is endothelium-dependent. Gui et al. observed that Up₄A at a concentration range of 10^–6–10^–5 mmol/L induced a relaxation response in SD rats whose pulmonary arteries were preliminarily relaxed using acetylcholine [19]. However, Up₄A did not induce a relaxation response in pulmonary arteries of SD rats whose endothelia were denuded.

This suggests that endothelium is required for Up₄A-mediated pulmonary vasodilation and that P2Y₂/P2Y₄ receptors are implicated in this Up₄A-mediated relaxation response. The pleiotropic vascular effects of Up4A appear to be contingent on the types of receptors activated and the consequent downstream signaling pathways. The work of Matsumoto adds another dimension to the story of Up₄A -mediated vasodilation [20]. In the aorta cycle of LETO rats, not only did Up₄A induce vasodilation, but this vasodilatory effect could be inhibited by both a selective COX1 inhibitor (VAS) and a COX2 inhibitor (NS39). This sheds light on the potential involvement of the COX₁-TXS-TxA2 endothelial pathway in mediating the vasodilatory action of Up₄A. Further complexity emerges from the studies conducted by Zhou et al. [21, 22] Their findings unveiled that the vasodilatory effect of Up4A on coronary arterioles, which were previously constricted using U46619 (100 nmol/L), could be inhibited by both the P2Y₆ receptor blocker (MRS2578) and the P2Y1 receptor blocker (MRS2179). These observations accentuate the roles of the P2Y₁ and P2Y₆ receptors in mediating Up₄A -induced vasodilation. Taking into account of these findings, it becomes evident that Up₄A exerts a dual role in vascular modulation: it induces vasoconstriction predominantly through P2X₁ receptors, while it prompts vasodilation primarily via P2Y-related receptors. The simultaneous activation of these opposing pathways may underlie the biphasic blood pressure response—both a decrease and a subsequent increase—observed in anesthetized mice upon a single injection of Up₄A via the tail vein in this experiment. This intricate balance between vasoconstriction and vasodilation, governed by the interaction of Up₄A with specific receptors, underscores the potential pharmacological significance of the compound and necessitates further exploration in both physiological and pathological contexts.

To encapsulate, Up₄A presents a unique and complex profile in the regulation of blood pressure. Contrasting sharply with the effects elicited by the P2X₁ receptor agonist—α, β-meATP and the α1 receptor agonist—NA, Up₄A administration results in a conspicuous biphasic response. This begins with an initial drop in blood pressure, followed by an upswing, albeit the surge is notably less pronounced than that triggered by α, β-meATP or NA at equivalent dosages.

A striking feature of the effect of Up₄A is the prolonged duration over which blood pressure returns to baseline levels. Moreover, the drug disproportionately impacts diastolic pressure over systolic pressure. Surveying existing literature and integrating the findings from this study, it becomes evident that this dual action—initial dilation followed by constriction—is ascribable to the combined effects of Up₄A on P2Y-related receptors, facilitating vasodilation, and P2X₁ receptors, mediating vasoconstriction. An important clinical observation to underscore is the elevated Up₄A levels in the plasma of adolescent patients with hypertension. This surge could be a consequence of vascular endothelial cells ramping up Up₄A secretion as a counteractive measure to attenuate escalating blood pressure. However, whether this compensatory mechanism inadvertently aggravates hypertension through heightened systemic resistance and vascular tone remains an open question. Future studies delving into this mechanism could have valuable therapeutic implications, offering fresh avenues to target the insidious progression of hypertension.

However, there are some limitations in the present study. We will consider to use P2Y and P2X receptor antagonists to block the effect of Up4A in the future, and make the work more impactful and increase its relevance and applicability.

Conclusion

Due to changes in administration method and route, the action of Up₄A on blood pressure may produce various experiment results. The administration of medication to mice through the superficial peripheral veins more closely resembles clinical conditions. A single injection of Up4A into the peripheral vein of anesthetized mice can elicit a biphasic change in the blood pressure of the common carotid arteries, characterized by a fall followed by an increase. After Up4A enters the blood arteries, it is hypothesized that the pyrimidine structure interacts to receptors associated with the vascular endothelium to produce a vasodilatory effect. In order to mediate vasoconstriction, the purine structure interacts to receptors associated with vascular smooth muscle. In this experiment, the blood pressure recovery time caused by Up₄A was longer compared to that induced by α, β-meATP or NA. Additionally, Up₄A influenced diastolic pressure more strongly than systolic pressure. Further research is needed to assess whether these phenomena exacerbate the progression of hypertension.