Heavy Alcohol Consumption Attenuates Human Mesenteric Artery Responsiveness to Sigma Receptor-1 Ligands

Abstract

Sigma receptor agonists are suspected to modulate blood pressure in humans. We investigated how modulation of sigma receptors impacts phenylephrine (PE)-induced contraction in human mesenteric arterial rings obtained from human organ donors. This study also explored the relationship between sigma receptor activation, PE-induced arterial contraction, and the history of the organ donor’s alcohol use. The concentration responsiveness of PE-induced arterial contraction was tested using wire myography in the absence and presence of the sigma receptor agonist PRE-084, and the sigma receptor antagonists BD-1047 and SM-21. Sigma receptor-1 expression in the arteries was also investigated using an automated capillary electrophoresis system. The results show that PRE-084 elicited a downward shift in the PE concentration-response curve. Notably, this trend only occurred in arteries from donors with histories of non-/light drinking or moderate drinking (P<0.05), but not with arteries obtained from donors with histories of heavy or binge drinking. The sigma receptor-1 antagonist BD-1047 elicited an upward shift in the PE concentration-response curve in arteries from non-/light and moderate drinkers, but not from heavy drinkers. Interestingly, the sigma receptor-2 antagonist caused an upward shift in the PE concentration-response curve in arteries from all three groups of donors. Notably, sigma receptor-1 protein levels were decreased in arteries from heavy drinkers compared to the other groups. Collectively, the findings suggest that sigma receptors in human arteries may promote relaxation. However, heavy alcohol consumption reduces arterial sigma receptor-1 expression and impairs its ability to modulate contraction.

NEW AND NOTEWORTHY

Activation or inhibition of sigma receptor-1 was found to modulate phenylephrine-induced contraction of isolated mesenteric arteries from human organ donors. However, this effect was impaired in arteries from donors who were heavy alcohol consumers, because the arteries from these individuals had relatively low protein expression of sigma receptor-1. These findings reveal a potential new role of sigma receptor-1 in the control of arterial tone in humans that is modulated by alcohol use.

INTRODUCTION

Alcohol misuse has been recognized as the primary risk factor for premature death and disability for people between the ages of 15 and 49 and is ranked fifth among all ages (1). One-fourth of total deaths in people between 20 and 39 years are attributed to alcohol misuse (2). Alcohol consumption has a complex relationship with cardiovascular health. It is suggested that moderate alcohol intake can be beneficial to cardiovascular health, while excessive consumption is linked to adverse outcomes, including hypertension, cardiomyopathy, and increased risk of stroke (3). In addition, there is strong evidence that consistent, heavy alcohol consumption is associated with higher cardiovascular risk (4, 5). Chronic alcohol exposure can lead to oxidative stress, inflammation, and impaired vascular function, contributing to the development of cardiovascular diseases (6). To provide better treatment for patients, it is essential to gain a more comprehensive understanding of the underlying mechanisms of alcohol-induced changes in cardiovascular function.

Sigma receptor-1 (σ₁) has been suggested as a potential pharmacological target for alcohol use disorder treatment. In various animal models, σ₁ antagonists reduce alcohol consumption, motivation to drink, and alcohol-seeking behavior, demonstrating a critical role for σ₁ in these behaviors (1, 7). The σ receptors are enigmatic proteins that were initially (and mistakenly) classified as a subtype of the opiate receptors to mediate the unique psychotomimetic effects of N-allylnormetazocine in 1967 (8). There are two primary isoforms in the σ receptor family σ₁ and σ₂ (9), which are expressed intracellularly in neurons and glial cells and modulate ion channel function. They reside in the endoplasmic reticulum and act as pharmacochaperones to regulate neurotransmission by affecting the expression level of a variety of different receptors and transporters (10). In the brain, σ₁ was reported to act like a “gain switch” that can amplify or reduce preference for alcohol in rodent models. While putative σ receptor agonists increase binge-like drinking behavior, σ antagonists produce the opposite effect (11-16). Notably, Sardinian alcohol-preferring rats were reported to have elevated σ₁ expression in the nucleus accumbens, a central part of the brain’s reward circuit, compared to alcohol-naïve outbred Wistar rats. The elevated σ₁ level became lower after a 4-week voluntary alcohol-drinking period, which was suggested as a possible mechanism of reduced motivation to drink following chronic drinking (11).

Currently, there is lack of knowledge in the understanding of the relation between alcohol consumption and vascular σ receptors, and there are few studies on how σ receptor dysregulation might contribute to the development of cardiovascular diseases. In our laboratory, we found that the selective σ₁ ligand PRE-084 reduces endothelial monolayer permeability (17). There is also a report claiming that afobazole, a σ agonist used in some countries in Europe to treat anxiety, may also normalize blood pressure in patients with hypertension (18). We have observed that afobazole causes relaxation of isolated rat lymphatic vessels (19), and formulated the hypothesis that σ receptors may also play a role in regulating vascular tone.

To test our hypothesis, we designed a study to evaluate the potential role of σ receptors in the regulation of arterial tone. For our model we used human mesenteric arteries isolated from organ donors and focused on how the σ₁ agonist PRE-084 affects contraction elicited by the α-adrenergic agonist phenylephrine. In addition, based upon initial observations that some arteries from human donors with history of heavy alcohol consumption were insensitive to PRE-084, we tested the hypothesis that chronic heavy alcohol consumption weakens the ability of σ₁ activation to limit adrenergic receptor-induced arterial contraction.

MATERIALS AND METHODS

Human Specimen Research

Viable human small intestine and mesentery was obtained from LifeLink^®, a Tampa-based nonprofit corporation that operates federally certified organ procurements and FDA/AATB certified tissue banks in recovering and processing human organs for transplantation. The organs were surgically removed and processed by designated transplant surgical teams at hospitals throughout Florida and transported via authorized medical carriers. Work with tissues from decedents does not meet the NIH definition of human subject research and no IRB approval was required. Nevertheless, donors were deidentified according to HIPAA and IRB standards by LifeLink prior to provision of medical history data to the University of South Florida (USF).

Tissue Collection and Preparation

Small intestines from organ donors (N=73) of both sexes with varied backgrounds, ranging in age from 7 to 82 years old, were included in the study. Blood was flushed and replaced with cold UW organ preservation solution prior to organ removal and storage on ice. Organs were transported on ice from hospitals to LifeLink’s Tampa Bay facility and then to USF. Total cold ischemia time, defined operationally as the time of perfusion to the initiation of procedures, ranged from 3.5 to 23.5 hours.

Upon arrival at the laboratory, the duodenum and proximal jejunum (to about 30 cm from the Sphincter of Oddi) and associated mesentery were dissected from the remainder of the intestine. Segments were placed on a 10-cm square tray in ice-cold albumin physiological salt solution (APSS: NaCl, 120 mM; KCl, 4.7 mM; CaCl₂·2H₂O, 2 mM; MgSO₄·7H₂O, 1.2 mM; NaH₂PO₄, 1.2 mM; Na pyruvate, 2 mM; glucose, 5 mM; EDTA, 0.02 mM; MOPS, 3 mM and purified BSA 1 g/100 ml). The ends of the intestine were clamped prior to excision to prevent luminal contents from escaping. Fourth or fifth order mesenteric arteries were dissected by first excising an artery/vein pair starting on the intestinal wall and extending approximately 5 cm into the mesentery. The excised segments were transferred to a small round dish filled with cold APSS, where they were carefully cleaned by removing fat cells and surrounding tissue, using blunt dissection to isolate at least 2 cm of artery. The cleaned segments were then cut into rings of 2 mm length and mounted onto parallel wires of the DMT 620M Wire Myograph system (DMT-USA, Ann Arbor, MI), submerged in 5 ml of cold APSS. The rings typically had an internal diameter greater than 500 μm, so 200 μm wires were used for all the experiments. Eight artery rings were obtained from each donor and measured simultaneously in eight myograph chambers. This allowed all sigma agonists and antagonists to be applied and duplication of drug protocols for each donor.

Experimental Protocol

The arterial rings in the DMT wire myograph were gradually warmed to 37 °C and allowed to equilibrate for at least 30 min. Tension of the arterial rings was recorded using LabChart 8.1.20 software (AD Instruments, Colorado Springs, CO). The rings were subjected to an optimal stretch simulating a transmural pressure of 13.3 kPa using the DMT normalization module and adjusting the separation of the myograph wires. After adjusting each vessel to its optimal diameter for studying tension, the vessels were allowed to stabilize for at least 30 min at 37 °C. Vessel viabilities were assessed by inducing contractions by elevating the extracellular K⁺ concentration, replacing 60 mM NaCl in the APSS bath solution with 60 mM KCl for 5 minutes. Arterial rings were considered viable if they developed robust tension (higher than 15 mN).

Following viability testing, the arteries were relaxed by washing 3 times with 5 ml warm PSS (NaCl, 118 mM; NaHCO₃, 25 mM; KCl, 4.7 mM; KH₂PO₄, 1.2 mM; MgSO₄·7H₂O, 1.17 mM; CaCl₂, 2.5 mM, glucose, 5.6 mM, pH 7.4) followed by a return to 5 ml of warm APSS solution. After a return to a steady baseline, to test the role of sigma receptors in modulating phenylephrine (PE)-induced contraction, vessels were first treated with either PBS (vehicle control, Gibco 10010-023, ThermoFisher, Waltham, MA), 100 μM PRE-084 (σ₁ agonist, Tocris, Minneapolis, MN), 200 nM BD-1047 (σ₁ antagonist, Tocris), or 200 nM SM-21 (σ₂ antagonist, Tocris) for 5 min. The concentrations for these drugs were based upon previous work. PRE-084 was previously shown to enhance barrier function of endothelial monolayers, with a half-maximal protective effect near 50 μM and its greatest effect in the 150-200 μM range (17, 20). Previous studies utilizing BD-1047 frequently use concentrations as high as 10 μM to block σ₁ (21, 22), but we previously found that using only 200 nM BD1047 attenuates the ability of the non-selective σ agonist afobazole to cause lymphatic vessel relaxation (19). The K_i for SM-21 binding to σ₂ is 145 ± 7 nM and ~ 1 μM for σ₁ (23), so we chose a concentration of 200 μM for this study. After the addition of PRE-084, BD-1047, or SM-21, the α₁-adrenergic agonist PE was added in serial, increasing concentrations: 100 nM, 300 nM, 1 μM, 3 μM, 10 μM, 30 μM, 100 μM, and 300 μM, with time allowed between to allow for contraction to develop and plateau. The time between each concentration was typically 5 min. Throughout the experiment, the tension-time curve displayed by LabChart was monitored, ensuring proper timing for the addition of substances and stability of tension levels.

After the final PE concentration was tested, artery rings were exposed to acetylcholine (ACh) to test endothelial-dependent relaxation. ACh was added in increments: initially at a 1 μM final concentration, allowing a 5-minute incubation period. If little to no relaxation was detected with 1 μM ACh, then 10 or 100 μM ACh was tested. At the end of the protocol, the ability to contract in response to 60 mM KCl was again tested to ensure viability was the same level as at the beginning of the experiment. Additionally, 1 μM sodium nitroprusside (SNP) was added during the plateau phase of the 60 mM KCl-induced contraction to evaluate the ability of the smooth muscle to relax in the presence of NO.

The software provided real-time feedback, allowing the researcher to determine the appropriate timing for adding subsequent substances. Logical assessments were conducted throughout the experiment to confirm the vessels' functional state. Following the wire myograph procedure, arterial rings were frozen and stored at −80°C for further biochemical analyses.

Assessment of Sigma Receptor Expression in Arteries

The protein levels of σ₁ in human mesenteric arteries were determined using the Protein Simple WES capillary western blot system (San Jose, CA, USA) according to manufacturer’s instructions. Briefly, frozen artery segments were thawed and homogenized in 250 μl ice-cold RIPA buffer (Millipore Sigma, Burlington, MA) containing HALT protease/phosphatase inhibitors (ThermoFisher), using a BeadBug homogenizer and tubes containing 3.0 mm zirconium beads (ThermoFisher). Homogenization was performed using three 30-s pulses at 4000 RPM and placing the tubes in ice for 1 min between each pulse. The lysates were then centrifuged at 14000 x g to remove large debris. Protein concentrations were determined using the BCA protein assay (ThermoFisher).

For the WES assay, the samples were diluted to a total protein concentration of 0.2 mg/ml. Lysates that had an initial protein concentration lower than this could not be used and were discarded, leaving a total of N=34 samples available for analysis. A rabbit polyclonal anti sigma-1R antibody (NBP1-82479, NovusBio, Centennial, CO) was used to detect σ₁, and a mouse monoclonal anti-β-actin antibody (#3700, Cell Signaling Technology, Boston, MA) was used to detect actin, which served as a loading control for normalization. The specificity of the anti-sigma-1R antibody was previously validated in two different publications in which siRNA-mediated knockdown of σ₁ significantly reduced the band intensity at ~25 kDa (17, 20). Both antibodies were used at a 1:50 dilution. Anti-rabbit and anti-mouse secondary antibodies were supplied with the WES detection module kits from Protein Simple. The assay was run and analyzed using ProteinSimple Compass software. The software generated images of lanes and bands with detected proteins based upon the densitometry data. The raw densitometry peaks were utilized for quantitative analyses.

Immunofluorescence Labeling and Microscopy

Small explants of human mesenteric tissue were excised from the intestine of two donors (an 18-year-old male and a 67-year-old female). Each tissue was transferred to a dish containing ice-cold physiological salt solution without albumin. Excess adipose tissue was carefully removed with forceps, leaving minimal residual fat. A 0.5-cm thick section of mesenteric tissue was excised and placed in a 15-mL sterile tube containing 10 mL of 10% neutral-buffered formalin (NBF) and fixed for either 24 hours at 4 °C or 1 hour at room temperature. Following fixation, tissues were rinsed three times in 10 mL sterile water, dehydrated through a graded ethanol series (70%, 80%, 95%, 100%) for 30 minutes each at room temperature, and cleared twice in xylene for 1 hour each at 4 °C. Dehydrated tissues were immersed twice in paraffin, briefly changing between immersions, and then embedded either vertically or horizontally. Paraffin blocks were sectioned at 5-μm thickness using a microtome, and sections were mounted on pre-cleaned glass microscope slides, which were baked at 37 °C for 10 minutes and stored at room temperature until staining. For staining, slides were deparaffinized by two 5-minute washes in xylene, rehydrated through a graded ethanol series (95%, 70%, 50%), and rinsed twice in distilled water for 3 minutes each. Antigen retrieval was performed by incubating slides in 300 mL of 1× sodium citrate buffer (pH 6.0) at 110 °C for 9 minutes in a pressure cooker, followed by cooling at room temperature for 20 minutes and two rinses in 1× TBS (5 minutes each). Blocking solution was prepared by combining 12.5 μL Triton X-100, 0.5 g BSA, and 1.12 g of reagent (clarification needed) in 40 mL of 1× TBS. A PAP pen was used to encircle the tissue, and slides were incubated in blocking solution at room temperature for 1 hour. Primary antibodies were diluted in 1× TBS containing 0.025% Triton and 1% BSA, and slides were incubated overnight at 4 °C. The primary antibodies (and final concentrations) used were goat anti-CD31 (R&D Systems AF3628, 10 μg/ml), rabbit anti-sigmaR1 (NovusBio NBP1-82479, 3.4 μg/ml), and mouse anti-alpha-smooth muscle actin conjugated to AlexaFluor 594 (R&D Systems IC1420T, 1 μg/ml). The following morning, slides were washed four times in 1× TBS for 5 minutes each and then incubated with the secondary antibodies donkey anti-rabbit AlexaFluor-488 and donkey anti-goat AlexaFluor-647 (Thermo A-21206 and A-21447, both applied at 1:500) for 1 hour at room temperature. Afterward, the tissue was washed three times in 1× TBS for 5 minutes each, and excess liquid was removed. ProLong Gold antifade reagent with DAPI was applied to each slide, followed by coverslip placement. Slides were placed vertically on filter paper to drain excess mounting medium and allowed to dry in the dark for 24 hours at room temperature. Images were obtained using an Olympus BX53 microscope system.

Data Collection and Analysis:

Donor characteristics (age, sex, weight, medical history) were provided by LifeLink and entered into a Microsoft Access database. Each donor also had a unique ID number. The medical histories included details about individual alcohol consumption levels. The donors were organized into alcohol consumption groups based upon definitions established by NIAAA (24). For men, heavy drinking is defined as consuming more than 15 drinks per week or more than 5 drinks at least once a week, while for women, heavy drinking is defined as consuming more than 8 drinks per week or more than 4 drinks at least once a week. Moderate drinkers included men who consumed up to two drinks per day and women who consumed one drink per day (25). Light drinkers encompassed individuals who consumed alcohol below these thresholds. Light drinkers were grouped together with nondrinkers, which served as the control group.

For the wire myograph protocol data, 1-min periods of tension data at baseline and during the period of the maximal change in tension after addition of test compounds were recorded for the data analysis. For ACh-induced relaxation, the baseline tension before PE addition (Baseline), the tension just prior to addition of ACh (Max) and the minimum tension measured within 10 min after ACh addition (Min) were used to calculate % Relaxation: $100\%\times (\text{Max}-\text{Min})∕(\text{Max}-\text{Baseline})$ These data were exported in Microsoft Excel format and saved with the corresponding donor ID number and date in the file name for easy reference and traceability. At the time of each experiment, the investigators were blinded to the medical histories of the donors.

After each donor in the entire cohort was classified into an alcohol consumption group (heavy, moderate, or light/nondrinker), the medical history data was combined with the wire myograph data. This was done in an Excel spreadsheet used to segregate the data into the alcohol consumption categories using separate tabs for each group. The data from each group was then transcribed into GraphPad Prism 10.3.1 software for statistical analysis. Concentration-response curves for PE in both the presence and absence of PRE-084, BD-1047, or SM-21 were generated for each group using the log(agonist) vs. response (three parameters) model in Prism, which utilizes the equation $\mathrm{Y}=\text{Bottom}+(\text{Top-Bottom})∕(1+{10}^{\wedge }({\mathrm{LogEC}}_{50}\text{-}\mathrm{X})$ Data points in each curve are presented as the mean ± standard error (SE). The differences in results for PRE-084, BD-1047, or SM-21 versus vehicle for each group (light/nondrinkers, moderate, and heavy drinkers) were analyzed with two-way ANOVA followed by a Sidak's correction multiple comparisons test to evaluate differences at each concentration. In addition, the curve characteristics (EC₅₀ and Maximal Tension) were compared for each group using an Extra Sum-of-Squares F test. Significance was accepted at P<0.05.

For σ₁ protein expression levels detected by WES, the peak areas for σ₁ were divided by the peak areas for β-actin, and these ratios are expressed as means ± SE for each group. Because the data did not follow a normal distribution, the nonparametric Kruskal-Wallis test was used for initial comparisons of the three alcohol groups, followed by Dunn’s multiple comparison test. Statistical significance was defined as P<0.05.

RESULTS

PRE-084 Reduces PE-Induced Arterial Contraction.

To evaluate the how PRE-084 pretreated human mesenteric arteries response to the α₁ receptor agonist PE, we performed a series of myograph experiments. Two example traces of artery tension vs. time from the experimental protocol are shown in Fig. 1. The protocol started with an arterial viability check with high potassium bath solution, followed by washout and re-establishment of baseline tension. Afterward, either vehicle (Fig. 1A), or PRE-084 (Fig. 1B) was added to the bath, followed by serial additions of PE at increasing concentrations that were used to construct a concentration-response relationship. After the last concentration of PE was added, endothelial-dependent relaxation was tested using ACh, followed by a washout. At the end of the protocol, a second viability test with high potassium was performed, immediately followed by a test of the ability of the smooth muscle layer to relax in response to sodium nitroprusside (SNP). Vessels were considered acceptable for inclusion in the study if they displayed contraction in response to high potassium, relaxation in response to SNP, and relaxation in response to ACh (provided there was sufficient contraction in response to PE for the ACh test to be meaningful).

Fig. 1.

Representative traces of the experimental protocol used to measure the concentration-response relationship for PE-induced arterial contraction in the absence (A) and presence of PRE-084 (B). The protocol starts with a viability test using 60 mM KCl, followed by washout. After re-establishing a steady baseline tension, PBS (vehicle control) or PRE-084 are added, followed by increasing concentrations of PE to determine the concentration-response relationship for PE-induced arterial contraction. Acetylcholine (ACh) is then applied to evaluate the degree of endothelial-dependent relaxation, followed by washout. A second viability test with 60 mM KCl is performed, followed by a test of sodium nitroprusside (SNP)-induced relaxation. The two traces shown are from two arterial rings derived from the same mesenteric artery.

Pretreatment of the arteries with 100 μM PRE-084 caused a downward shift in the PE concentration-response curve (Fig. 2A), indicated by a decrease in the maximal tension and no change in the EC₅₀ (Table 1). We also analyzed arteries from female donors (Fig. 2B) or male donors (Fig. 2C) separately. For females, arterial contraction elicited by 1, 3, 10, or 300 μM PE was lower in the presence of PRE-084 (Fig. 2B), while for males, this effect occurred with 1, 3, 10, 30, 100, and 300 μM PE (Fig. 2C). The maximal tension of the PE concentration-response curve was significantly lower (P<0.05) when PRE-084 was present for the males, but not for females, and for both sexes there was no significant change in the EC₅₀ (Table 1). Looking at the PE concentration-response curves of vehicle-treated arteries between females and males, there were no significant differences in tension at all the individual concentrations tested (Fig. 2D). However, in the curve-fit analyses, the maximal tension the PE concentration-response curve was found to be significantly higher for arteries from female donors than those from males while there was no difference in the EC₅₀ (Table 2). Overall, these data show that PRE-084 causes a downward shift in the concentration-response curve for PE-induced contraction in human mesenteric arteries. We also detected subtle differences between arteries from males and females.

Fig. 2.

PRE-084 decreases PE-induced contraction of human mesenteric artery rings. Concentration-response relationships for PE-induced contraction in the absence and presence of 100 μM PRE-084 are shown for (A) all donors studied, (B) female donors, and (C) male donors. Panel D shows the concentration response relationship in the absence of PRE-084, comparing male and female donors. *P<0.05 between groups for the concentration indicated on the graph. Data was analyzed using two-way, repeated-measures ANOVA, followed by Sidak’s post-hoc test for multiple comparisons. The number of subjects (donors) in each group are shown on each panel.

Table 1.

Impact of PRE-084 of PE-Concentration-Response Curve Parameters.

	Maximum Tension (mN)			EC50 (μM)
	Vehicle	PRE-084	P-value	Vehicle	PRE-084	P-value
All Donors	12.94	10.56	0.0020*	0.899	1.804	0.2583
Females	15.94	12.88	0.0611	0.950	2.154	0.3922
Males	11.51	9.35	0.0084*	0.918	1.632	0.4780

Table 2.

Comparison of PE-Concentration-Response Curve Parameters Across Sexes.

	Maximum Tension (mN)			EC50 (μM)
	Females	Males	P-value	Females	Males	P-value
Vehicle	15.94	11.51	0.0020*	0.950	0.918	0.9690

Heavy Alcohol Consumption Alters the Impact of PRE-084 on PE-Induced Arterial Contraction.

We noticed that arteries from some donors displayed no difference in their PE concentration-response curves with PRE-084 treatment compared to vehicle control. These arteries tended to come from donors with histories heavy alcohol consumption, so we compared the ability of PRE-084 to alter PE-induced contraction of human mesenteric arteries based upon alcohol consumption history. Table 3 shows the donor characteristics for the three groups. There were no significant differences in age, height, weight, or sex among the groups, or with other characteristics shown.

Table 3.

Donor Characteristics Across the Alcohol Consumption Groups.

Drinking Level	Non-/Light	Moderate	Heavy
Number of donors	32	21	20
Women	14 (44%)	7 (33%)	6 (30%)
Mean Age ± S.D.	49.1 ± 16.6	44.9 ± 14.3	48.0 ± 14.6
Mean Height ± S.D.	49.1 ± 4.8	67.2 ± 3.4	68.0 ± 4.6
Mean Weight ± S.D.	94.6 ± 46.9	83.9 ± 14.5	94.1 ± 27.8
Mean Calculated BMI ± S.D.	29.7 ± 7.2	28.8 ± 5.0	29.7± 4.9
Race/Ethnicity
White	19 (59%)	15 (71%)	17 (85%)
Black/African American	3 (9%)	1 (5%)	0 (0%)
Hispanic/Latino	10 (31%)	5 (24%)	3 (15%)
History of Smoking	16 (41%)	13 (62%)	15 (75%)
History of Illicit Drug Use	13 (41%)	13 (62%)	11 (55%)
Hypertension	8 (25%)	5 (24%)	5 (25%)
Diabetes	6 (19%)	2 (10%)	2 (10%)

For the control group, PRE-084 caused a downward shift in the PE concentration-response curve, with significant differences at 3, 10, 30, 100, and 300 μM PE (Fig. 3A) and a decrease in the maximal tension with no change in the EC₅₀ (Table 4). A similar downward shift in the presence of PRE-084 was also apparent for the moderate alcohol consumption group, with significant differences at 1, 3, 10, 30, 100, and 300 μM PE (Fig. 3B), and a significantly decreased maximal tension with no change in the EC₅₀ (Table 4). However, in the heavy alcohol consumption group, PRE-084 did not shift the concentration-response curve for PE, with no significant differences among individual data points or between curve parameters (Fig. 3C) and no change in the maximal tension or EC₅₀ (Table 4). This suggests that heavy alcohol consumption impairs the ability of PRE-084 to decrease human mesenteric artery contraction in response to PE.

Fig. 3.

Heavy alcohol use diminishes the impact of PRE-084 on PE-induced arterial contraction. Concentration-response relationships for PE-induced contraction in the absence and presence of 100 μM PRE-084 are shown for donors classified as (A) non-/light drinkers, (B) moderate drinkers, and (C) heavy drinkers of alcohol. *P<0.05 between groups for the concentration indicated on the graph. Data was analyzed using two-way, repeated-measures ANOVA, followed by Sidak’s post-hoc test for multiple comparisons. The number of subjects (donors) in each group are shown on each panel.

Table 4.

Alcohol Consumption Levels and the Impact of PRE-084 of PE-Concentration-Response Curve Parameters.

	Maximum Tension (mN)			EC50 (μM)
	Vehicle	PRE-084	P-value	Vehicle	PRE-084	P-value
Non-/Light	13.44	10.54	0.0238*	1.258	1.929	0.6652
Moderate	12.75	9.05	0.0409*	0.737	1.668	0.6376
Heavy	13.04	12.12	0.4328	0.704	1.913	0.2320

Also, we tested endothelial-dependent relaxation in arteries that had sufficient sustained contraction in response to PE. While the mean response to ACh in the moderate alcohol consumption group appears noticeably lower than the non/light and heavy alcohol consumption groups, the means were not significantly different due to the wide variation in responses (Fig. 4). Importantly, the heavy alcohol consumption group had equivalent ACh-induced relaxation to that of the non/light group, indicating that ACh-induced endothelial-dependent relaxation does not appear to be impaired in the arteries from heavy alcohol users.

Fig. 4.

ACh-induced relaxation of PE-constricted arteries. The % Relaxation was calculated as the change in tension after addition of ACh in comparison to the tension just prior to the addition of ACh. Only arteries that had sustained contraction after addition of PE were used for analysis. The number of subjects in each group were: Non/Light, N=10; Moderate, N=9; Heavy, N=6. The alcohol consumption groups were compared by one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. There were no significant differences between groups.

Alcohol Consumption Impairs the Impact of a σ₁ Antagonist on PE-Induced Arterial Contraction.

After the initial observations of how PRE-084 impacted the concentration-response curve for PE, we decided to test whether a σ₁ antagonist affects PE-induced contraction and whether alcohol consumption levels modulate their effects. Opposite to that of PRE-084, application of the σ₁ antagonist BD-1047 caused an apparent upward shift in the PE concentration-response curve, with the tension elicited by 30 μM PE was significantly higher in the presence of BD-1047 than in its absence (Fig. 5A). The maximal tension of the concentration-response curve was significantly higher in the presence of BD-1047, while the EC₅₀ was not significantly changed (Table 5). Examining how alcohol consumption impacts the relationship, the upward shift in the concentration-response curve elicited by BD-1047 was maintained in the non-/light and moderate drinkers (Fig. 5B-C), with both groups having elevated maximal tension (Table 5). However, for heavy drinkers the curves were similar in the presence and absence of BD-1047 (Fig. 5D) and there was no difference in maximal tension (Table 5). The data suggest that treatment of human mesenteric arteries with the σ₁ antagonist BD-1047 increases the force of PE-induced contractions. However, heavy alcohol consumption diminishes the ability of BD-1047 to enhance PE-induced contraction.

Fig. 5.

BD-1047 enhances PE-induced arterial contraction in non-/light and moderate users of alcohol. Concentration-response relationships for PE-induced contraction in the absence and presence of 200 nM BD-1047 are shown for (A) all donors, (B) non-/light drinkers, (C) moderate drinkers, and (D) heavy drinkers of alcohol. *P<0.05 between groups for the concentration indicated on the graph. Data was analyzed using two-way, repeated-measures ANOVA, followed by Sidak’s post-hoc test for multiple comparisons. The number of subjects (donors) in each group are shown on each panel.

Table 5.

Impact of BD-1047 on the PE-Concentration-Response Curve Parameters for All Donors and Across Alcohol Consumption Groups.

	Maximum Tension (mN)			EC50 (μM)
	Vehicle	BD-1047	P-value	Vehicle	BD-1047	P-value
All Donors	12.02	19.07	<0.0001*	1.068	1.801	0.5484
Non-/Light	10.14	16.72	0.0019*	1.189	1.605	0.8226
Moderate	15.29	27.45	0.0195*	1.284	2.607	0.6820
Heavy	12.55	14.13	0.3728	0.754	0.969	0.8266

Alcohol Does Not Impact the Effect of a σ₂ Antagonist on PE-Induced Arterial Contraction.

We also studied how the σ₂ antagonist SM-21 may affect PE-induced arterial contraction and whether there was any impact from alcohol consumption history. SM-21 caused an upward shift in the PE concentration-response curve with a significantly elevated tension elicited by 100 μM PE (Fig. 6A) and a significantly elevated maximal tension with no significant change in the EC₅₀ (Table 6). When the data were grouped according to alcohol intake, the upward shift and significant elevation in the maximal tension of the PE concentration response-curve caused by SM-21 pretreatment persisted (Fig. 6B-D and Table 6). These findings suggest that the σ₂ antagonist SM-21 enhances PE-induced arterial contraction force. For the all the alcohol consumption groups, while some of the data points of the PE versus PE + SM-21 curves might appear different (especially for moderate alcohol consumers, Fig. 6C), these were not found to be significantly different when using Sidak’s multiple comparison test. However, there is a significant difference in the maximum tension calculated from each group’s concentration-response curve between PE alone and PE in the presence of SM-21 (Table 6), suggesting that the enhancement of PE-induced contraction in the presence of SM-21 is not impacted by alcohol intake.

Fig. 6.

SM-21 enhances PE-induced arterial contraction, regardless of alcohol use history. Concentration-response relationships for PE-induced contraction in the absence and presence of 200 nM SM-21 are shown for (A) all donors, (B) non-/light drinkers, (C) moderate drinkers, and (D) heavy drinkers of alcohol. *P<0.05 between groups for the concentration indicated on the graph, or between groups in the table. Data was analyzed using two-way, repeated-measures ANOVA, followed by Sidak’s post-hoc test for multiple comparisons. The number of subjects (donors) in each group are shown on each panel.

Table 6.

Impact of SM-21 on the PE-Concentration-Response Curve Parameters for All Donors and Across Alcohol Consumption Groups.

	Maximum Tension (mN)			EC50 (μM)
	Vehicle	SM-21	P-value	Vehicle	SM-21	P-value
All Donors	14.33	19.04	<0.0001*	0.545	1.525	0.0661
Non-/Light	13.31	16.53	0.0424*	0.641	1.390	0.3824
Moderate	16.09	24.32	0.0009*	0.569	2.509	0.1914
Heavy	14.29	18.19	0.0150*	0.395	1.092	0.2437

Heavy Alcohol Use Reduces σ₁ Expression in Human Mesenteric Arteries.

Because we observed that heavy alcohol consumption diminished the ability of both PRE-084, a σ₁ agonist, and BD-1047, a σ₁ antagonist, to respectively decrease and enhance PE-induced arterial contraction, we investigated if there is a relationship between alcohol consumption and σ₁ expression levels in human mesenteric arteries. Representative lane images generated by the WES instrument for σ₁ protein detection are shown in Fig. 7A. β-actin was also detected in the same lysates and served as a loading control (Fig. 7B). The band intensities of σ₁ protein normalized to β-actin as shown in Fig. 7C. We found that the arterial σ₁ protein levels were highest in the non-/light drinking group and significantly decreased in arteries from heavy drinkers. We also investigated the general localization of σ₁ using immunofluorescence microscopy of human mesenteric tissue sections featuring an artery. Representative results are shown in Fig. 8, and a mesenteric artery is denoted by the ‘a’ in Fig. 8A. Vessels were identified using antibodies to detect CD31, an endothelial cell marker (Fig. 8A), and smooth muscle actin to identify vascular smooth muscle cells (Fig. 8B). Sigma receptors were also labeled (Fig. 8C) and a composite image was constructed to visualize its overlap of the CD31 and SMA signals (Fig. 8D). In the larger artery, σ₁ labeling partially overlapped with CD31 and appeared to be on the basal side of endothelial cells, in a similar fashion as previously observed in rat lymphatic vessels (19). The results suggest that the decreased σ₁ protein levels observed in arteries from the heavy alcohol consumers is likely to be primarily in endothelial cells.

Fig. 7.

Expression of σ₁ is reduced in arteries from heavy alcohol users. A. Representative lanes showing (A) σ₁ and (B) β-actin protein detection from the WES instrument for non-/light, moderate, and heavy drinkers. Note that the lanes shown are separate images generated by the Compass software that controls the WES instrument that have been arranged into the three groups being compared. C. Quantitation of the peak areas for σ₁ divided by those for β-actin for all the samples studied. Because normal distributions were not observed, data were analyzed using the nonparametric Kruskal-Wallis test, followed by Dunn’s multiple comparison test. P-values for each comparison are indicated on the graph. The number of donor samples studied were N=15 for the non-/light drinker group, N=11 for the moderate drinker group, and N=13 for the heavy drinker group.

Fig. 8.

Localization of σ₁ expression in mesenteric tissue and vasculature. The images show a representative 5-μm section of mesentery from an 18-year-old male with no history of alcohol use, featuring an artery and surrounding tissue and vessels. The section was labeled to detect: A. CD31 (magenta), B. α-SMA (red), or C. σ₁ (green). D. Overlay image of CD31, α-SMA, and σ₁ including nuclei (blue). In panel A, ‘a’ denotes an artery and ‘v’ denotes a nearby vein. Scale bar = 100 μm and applies to all panels.

DISCUSSION

This study provides novel insights into the role of σ₁ activation on smooth muscle contraction in human mesenteric arteries and its potential modulation by alcohol consumption. First, the novel observation that PRE-084 causes a downward shift in the PE concentration-response curve suggests that σ₁ activation attenuates α₁-adrenergic-mediated vasoconstriction. In addition, the upward shift caused by BD-1047 suggests that inhibition of σ₁ enhances α₁-adrenergic-mediated vasoconstriction. Our work also demonstrates a significant reduction in σ₁ expression from human donors with histories of chronic heavy alcohol consumption that may explain the functional differences in arterial tissue. Specifically, heavy drinkers showed reduced modulation of PE-induced contraction by PRE-084 or BD-1047, contrasting with significant σ₁-receptor-mediated effects observed in non-/light and moderate drinkers. These findings corresponded with σ₁ downregulation in the arteries derived from heavy alcohol users. These findings highlight a potential mechanistic link between alcohol-induced cardiovascular risks and the disruption of σ₁-mediated vascular regulation, providing a new avenue for understanding and addressing the vascular complications of chronic heavy alcohol use.

The current results align with existing literature highlighting a potential protective role of σ₁ in the cardiovascular system. Our finding that PRE-084 reduces PE-induced arterial contraction is very similar to those reported from a recent, similar experiment utilizing pulmonary arteries from male rats (26). In this previous study PRE-084 caused an apparent downward shift in the PE concentration-response curve, and the reduced contraction was attributed to a decrease in K_v1.5 channel activity (26). In another study utilizing a mouse model of methamphetamine-induced hypertension, genetic deletion of σ₁ or chronic inhibition with BD-1047 exacerbated the increase in blood pressure and resulted in a greater degree of vascular remodeling (27). In the same study, chronic activation of σ₁ with PRE-084 prevented methamphetamine-induced hypertension and vascular remodeling during the 6-week study period (27). Moreover, a post-hoc analysis of the phase IIa clinical trial of ANAVEX2-73, a σ₁ agonist under investigation for treatment of Alzheimer’s Disease, revealed that this drug caused an average 8.7-mmHg drop in systolic blood pressure in the 32 patients enrolled in the study (28). It is worth noting that in addition to vascular effects, σ₁ also has a cardioprotective role. Hearts from Sigmar1^−/− mice have abnormally shaped cardiomyocytes and impaired mitochondrial function, resulting in contractile dysfunction (29). Moreover, administration of the σ₁ agonist SA4503 has been shown to protect against atrial fibrillation by reducing sympathetic nervous system activity (27). We also found in the current study that mesenteric arteries from organ donors with histories of heavy alcohol consumption had diminished levels of σ₁ expression. It is worth noting that reduced SIGMAR1 mRNA and σ₁ protein expression were reported in lung samples from pulmonary hypertension patients and heart samples from patients with atrial fibrillation (26), and in rat models featuring these conditions (30, 31). In the current study we also examined σ₁ localization in arteries was primarily in the endothelium, with little to no expression in the smooth muscle layer. This finding was very similar to the localization pattern we observed for σ₁ in rat mesenteric lymphatic vessels in a previous study (19). The finding also suggests that the changes in σ₁ protein expression found in heavy alcohol consumers are likely due primarily to changes in endothelial σ₁. Our new findings with human mesenteric arteries expand the notion that σ₁ has a protective role in the cardiovascular system and together with the previous findings, suggest an important, previously unrecognized role for σ₁ in arterial function.

Our results also provide new insight that may be relevant to hypertension associated with alcoholism. Several previous studies have shown that chronic alcohol treatment causes increased contraction of isolated aorta and mesenteric arteries in response to adrenergic stimuli (32-35). Elevated expression of α₁ adrenergic receptors was reported as early as two weeks after the start of chronic alcohol consumption in male Wistar rats (36). However, there are also acute effects, as direct addition of alcohol or its metabolites to isolated rat mesenteric arteries from naïve male Sprague-Dawley rats caused a leftward shift of the EC₅₀ for phenylephrine-induced contraction in concentration-response studies (37). Most studies have been performed with only male rats, but one study reported that alcohol-treated males had enhanced phenylephrine-induced aortic constriction while alcohol-treated females did not (33). In contrast, arteries from female rats chronically treated with alcohol were reported by other investigators to be more sensitive to KCl-induced contraction than from male alcohol-treated counterparts (38). Deciphering the mechanisms of how alcohol increases risk for hypertension in humans is a complex problem due to the multiple physiological systems that are likely contributors, including the central and peripheral nervous systems, the heart, endothelial and smooth muscle cells of arteries, the kidneys, and endocrine system (39). One potential cause relevant to the current study may be elevated plasma norepinephrine levels, which have been reported in alcohol-dependent individuals (40). At the same time, however, the reported levels of hypertension in the current study were similar among our groups stratified according to alcohol intake. Also, the concentration-response curves for PE-induced arterial contraction among non-/light, moderate, and heavy drinkers in the absence of PRE-084 were not significantly different. The novel finding of reduced σ₁ expression in arteries from heavy alcohol users is likely to be just one small piece of a much larger puzzle.

Human mesenteric arteries provide as a physiologically relevant model for examining the vascular effects of chronic alcohol consumption that is more directly translatable to human health compared to animal models. By analyzing arterial function across a spectrum of alcohol consumption levels, we capture a range of potential effects that reflect real-world variability. However, inherent challenges exist in working with human donor tissues. The diversity in donor characteristics, including differences in age, sex, and underlying health conditions, introduces variability that may influence vascular responses and σ₁ expression independent of alcohol consumption. We cannot control the samples that become available, making it difficult to obtain an even distribution of characteristics like age, sex, and race. Additionally, reliance on medical history records introduces the potential for inaccuracies, as self-reported alcohol consumption and comorbidities may not always be precise, and the data relies heavily on medical providers to be thorough in questions and documentation. The demographic distribution of our cohort further limits broad applicability, as the majority of donors were Caucasian, restricting our ability to generalize findings across diverse racial and ethnic populations. If possible, future studies with a more balanced demographic representation and improved clinical data accuracy could enhance the robustness of these findings.

A future direction for this study will be to understand the mechanism by which σ₁ agonists elicit a downward shift in the concentration-response curve for PE-induced arterial contraction. In the current study we observed that: 1) σ₁ is expressed in the arterial endothelium but not smooth muscle, and 2) PRE-084 had reduced activity to inhibit PE-induced contraction in arteries from heavy alcohol consumers in association with decreased σ₁ protein expression. Thus, it appears likely that the mechanism is dependent upon endothelial cells. One or more of the endothelial derived signaling molecules could thus be potentially involved. These include nitric oxide (NO), prostacyclin or other prostaglandins, endothelial-derived hyperpolarizing factors, hydrogen peroxide, or hydrogen sulfide (41). Identifying which of these factors have a role in the mechanism will be a key are of future investigation.

Overall, the findings provide new evidence that σ₁ activation can modulate vascular function by attenuating α-adrenergic-mediated arterial smooth muscle contraction. However, chronic, heavy alcohol consumption reduces σ₁ expression in arteries, disrupting the ability of σ₁ agonists to attenuate α-adrenergic-induced vasoconstriction. This alteration in vascular response may contribute to the underlying mechanisms of alcohol-induced hypertension, further highlighting the impact of sustained alcohol consumption on cardiovascular health. Further research is necessary to delineate these mechanisms and explore potential therapeutic strategies to mitigate the vascular consequences of chronic alcohol consumption.

DATA AVAILABILITY

Data will be made available upon reasonable request.