Aging alters spontaneous and neurotransmitter-mediated Ca²⁺ signaling in smooth muscle cells of mouse mesenteric arteries

Abstract

Objective

Aging impairs MA dilation by reducing the ability of sensory nerves to counteract sympathetic vasoconstriction. This study tested whether altered SMC Ca²⁺ signals to sympathetic (NE) and sensory (CGRP) neurotransmitters underlie aging-related deficits in vasodilation.

Methods

MAs from young and old mice were pressurized and loaded with Fluo-4 dye for confocal measurement of SMC Ca²⁺ sparks and waves. Endothelial denudation resolved the influence of ECs. SMCs were immunolabeled for RyR isoforms and compared to transcript levels for RyRs and CGRP receptor components.

Results

SMCs from young vs. old mice exhibited more spontaneous Ca²⁺ spark sites with no difference in Ca²⁺ waves. NE reduced spark sites and increased waves for both groups; addition of CGRP restored sparks and reduced waves only for young mice. Endothelial denudation attenuated Ca²⁺ responses to CGRP for young but not old mice, which were already attenuated, suggesting a diminished role for ECs with aging. CGRP receptor expression was similar between ages with increased serum CGRP in old mice, where RyR1 expression was replaced by RyR3.

Conclusion

With aging, we suggest that altered RyR expression in SMCs contributes to impaired ability of sensory neurotransmission to restore Ca²⁺ signaling underlying vasomotor control during sympathetic activation.

Introduction

Calcium signals mediate smooth muscle cell (SMC) contraction and relaxation, arterial diameter and tissue blood flow. In vascular SMCs, Ca²⁺ sparks entail a transient, localized increase in [Ca²⁺]_i that arises from the opening of ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) in response to increased cytosolic¹ or SR² [Ca²⁺] which activates nearby large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in the plasma membrane. These spontaneous transient outward currents (STOCs) hyperpolarize SMCs and promote vasodilation by inhibiting L-type voltage-gated Ca²⁺ channels (VGCCs)^3,4. Dysfunction in spark production and coupling to BK_Ca channels is evident in pathophysiological conditions including hypertension, diabetes and hemorrhagic shock⁵. In contrast to Ca²⁺ sparks, Ca²⁺ waves reflect increases in [Ca²⁺]_i throughout the cytoplasm arising from Ca²⁺- and inositol 1,4,5 trisphosphate (IP₃)-dependent activation of IP₃ receptors and/or RyRs. The activation of adjacent IP₃Rs and RyRs via calcium-induced calcium release (CICR) results in propagation of the Ca²⁺ wave throughout the cell⁶. Calcium sparks and waves occur with variable frequency under basal conditions in vascular SMCs^7–10 as affected by the expression and localization of RyR and IP₃R isoforms^7,11–13. Moreover, these signaling properties vary with vascular bed ⁵ and clinical pathology^14,15.

Aging has been shown to alter vascular SMC Ca²⁺ signaling via enhanced Ca²⁺ sensitivity, reduced CICR and slower refilling of SR Ca²⁺ stores^16,17,18,. A limitation of previous studies is their reliance on ratiometric measurements of fluorescent Ca²⁺ indicators such as Fura-2 AM. As a consequence, the properties of Ca²⁺ signaling events within individual SMCs of aged vessels remain undefined. Because the expression profile of RyR isoforms RyR1, RyR2 and RyR3 affect the nature of Ca²⁺ signals⁵, disproportionate changes in their profile may contribute to age-related changes. In the rat cervical ganglia, aging was associated with a selective increase in RyR3¹⁹, an isoform with different properties than RyR1 and RyR2. For example, rather than contribute to their production,²⁰ RyR3 may prevent Ca²⁺ sparks via inhibition of RyR1 and RyR2 as shown by increased STOCs in cerebral artery SMCs from RyR3-deficient mice²¹. Aging increases RyR3 and decreases RyR1 mRNA expression in SMCs of mouse MAs²², but whether such changes in transcript levels translate to altered protein expression or channel function is unknown.

In the resistance vasculature, endothelial dependent hyperpolarization initiates SMC relaxation by inhibiting VGCCs complemented by the release of vasodilator autacoids²³. While both effects reflect Ca²⁺ signaling in endothelial cells (ECs), little is known of how the endothelium affects SMC Ca²⁺ signals during aging. Recent studies have highlighted altered Ca²⁺ signaling in endothelial tubes of mouse resistance arteries,^24,25 intact pressurized rat carotid arteries ²⁶ and MA networks controlling tissue blood flow¹⁷. While denudation has been integral to defining the role of the endothelium in vasomotor control,²⁷ how removal of the endothelium affects SMC Ca²⁺ signals in advanced age is unexplored.

Perivascular sympathetic and sensory nerves regulate vasomotor function and can modulate vascular Ca²⁺ signaling via neurotransmitter release to activate membrane receptors on SMCs and ECs²⁸. In MAs, the sympathetic neurotransmitter norepinephrine (NE) and the sensory neurotransmitter calcitonin gene-related peptide (CGRP) are physiological mediators of neurogenic vasoconstriction and vasodilation, respectively. Previous studies in rat mesenteric arteries have described increases in SMC Ca²⁺ waves following direct²⁹ and pharmacologically-simulated³⁰ sympathetic nerve activation. Although the nature of CGRP-mediated decreases in SMC [Ca²⁺]_i has been explored in rat coronary arteries and gerbil spiral modiolar arteries^31,32, there are no studies of how CGRP affects SMC Ca²⁺ signals in blood vessels of mice.

The density and function of perivascular nerves declines with advancing age³³. In mouse MAs, vasomotor responses to stimulation of perivascular sympathetic nerves and of sensory nerves are both diminished, as is reciprocal inhibition between respective sources of vasomotor control^34,35. While MA dilation to exogenous CGRP declines with advanced age³⁵ the underlying changes in signaling events that contribute to this functional deficit is undefined. Endothelial denudation or inhibition of autacoid production attenuated dilation to CGRP³⁵, suggesting that EC signaling may contribute to sensory nerve dysfunction with advanced age. The subcellular localization of CGRP receptor components is also altered in advanced age, with RAMP1 shifting from membrane to perinuclear in intact MA ECs ³⁵. A reduction in receptor number may further impair dilation to CGRP.

In light of age-related deficits in sympathetic vasoconstriction, sensory vasodilation, and their reciprocal inhibition in MAs of mice^34,35, the purpose of this study was to test the hypotheses that: (1) Spontaneous and neurotransmitter-induced Ca²⁺ signaling in SMCs is altered with aging; (2) Endothelial denudation alters neurotransmitter-evoked Ca²⁺ signaling in MA SMCs of young and old mice; and (3) altered protein expression of RyR isoforms and/or CGRP receptor components contribute to impaired vasodilation to CGRP with advanced age. We focused on defining Ca²⁺ signals occurring spontaneously under control conditions, during stimulation with NE to simulate sympathetic nerve activation, and during the subsequent addition of CGRP to simulate the simultaneous activation of sensory and sympathetic nerves.

Materials and Methods

Ethical Approval

All procedures were reviewed and approved by the Institutional Animal Care and Use Committee of The University of Missouri (protocol # 9112) and performed in accord with the Guide for the Care and Use of Laboratory Animals³⁶ in compliance with the animal ethics checklist of this journal.

Animal Care and Use

Experiments were performed on young (3–6 mo, 20–27 g) and old (24–28 mo, 20–38 g), male C57BL/6J mice bred at colonies maintained by the National Institutes of Aging at Charles River Laboratories (Wilmington, MA). Respective age groups correspond to humans in their mid-20s and mid-60s³⁷. Mice were acclimated at the University of Missouri animal care facilities at least 1 week prior to study. Mice were maintained under a 12:12 h light/dark cycle at 22–24°C with fresh water and food available ad libitum. On the day of an experiment, a mouse was anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg kg⁻¹). Following tissue removal, the anaesthetized mouse was euthanized by cardiac puncture and exsanguination.

Vessel preparation and cannulation

The intact mesentery and small intestine were dissected free and placed in chilled (4°C, pH 7.4) Ca²⁺-free physiological salt solution (PSS) containing (in mM): 140 NaCl, 5 KCl, 1 MgCl₂, 10 HEPES, 10 Glucose³⁵. A jejunal segment was spread and pinned onto a block of clear silicone rubber (Sylgard 184, Dow Corning; Midland, MI; USA) and maintained at 4°C. Second-order MAs were dissected by hand while viewing through a stereomicroscope. For studies of endothelium-denuded MAs, a tungsten wire (diameter, 50 μm) was passed through the lumen 3 times to disrupt ECs while preserving the integrity of SMCs³⁵. Individual MAs were transferred to the cannulation chamber (RC-27N; Warner Instruments; Hamden, CT; USA) using a 50 μL Wiretrol II pipette (Drummond Scientific, Broomall, PA). The tissue chamber was mounted onto an aluminum platform containing 3-way micromanipulators (DT3–100; Siskiyou Design; Grants Pass, OR; USA) at each end for securing micropipettes. An isolated MA was cannulated at each end onto glass micropipettes [tip outer diameter (OD), ~100 μm] pulled (P-97; Sutter Instruments; Novato, CA; USA) from borosilicate glass capillaries (internal diameter (ID) = 0.94 mm; OD = 1.2 mm; Warner) with heat-polished tips and secured with a single strand from braided 10–0 silk suture³⁵. The aluminum platform was then positioned on the stage of a microscope (BX51W1; Olympus America Inc.; Center Valley, PA; USA) and the MA was pressurized to 100 cm H₂O. MAs were studied with constant superfusion of PSS (~5 ml min⁻¹) through the tissue chamber and without luminal flow. In each experiment, n represents the number of individual vessels with no more than 2 vessels used from each mouse.

Confocal imaging and analysis of SMC Ca²⁺ signals

Each MA was loaded with the Ca²⁺-indicator dye Fluo-4 AM (Cat. #F14201; ThermoFisher Scientific; Waltham, MA, USA) for 1 h by incubating (10 μM in Ca²⁺-free PSS) at room temperature. Following dye loading, vessels were superfused with PSS containing 2 mM CaCl₂ (referred to as control PSS) at 36°C and allowed to equilibrate for 30 min. Superfusion at 36°C was maintained throughout experiments via a temperature-controlled (SW-60; Warner) 20 mL reservoir fed continuously by a recirculating supply of PSS in the absence and presence of NE [167 or 600 nM; respective EC₅₀ values for MAs of young and old mice³⁵, CGRP [100 nM, a high concentration of CGRP at which vasodilation was impaired with aging ³⁵] or both. For fluorescence imaging, Fluo-4 was excited at 491 nm using a Cobalt Calypso laser (Cobalt Inc., Solna, Sweden). Each field of view (FOV) encompassed an area of 390 × 292 μm and contained ~15 SMCs. Images were acquired through a 20X water immersion objective (numerical aperture, 0.5) at 30 frames/s using a spinning disc confocal scanner unit (CSU-X1; Yokogawa, Tokyo, Japan) and CCD camera (XR-TURBO EX 620; Stanford Photonics, Palo Alto, CA, USA). To avoid laser-induced damage to vessel segments across multiple measurements, criterion recordings were limited to 10 s per treatment. Images were acquired using the same laser power and gain settings for both age groups using Piper Control Software (Stanford Photonics).

Analyses of SMC Ca²⁺ sparks and waves were performed in all cells using SparkAn software provided by Dr. Adrian Bonev (University of Vermont). For analysis of Ca²⁺ sparks, a 10 × 10 pixel region of interest (ROI; corresponding to an area of 6.25 × 6.25 μm²) was placed over each site exhibiting a Ca²⁺ spark during the recording period applying a boxcar average of 3 frames. Events with amplitudes greater than 1.2 F/F_o (where F = signal and F_o = baseline fluorescence) were analyzed. For Ca²⁺ waves, a 20 × 40 pixel ROI (corresponding to an area of 12.5 × 25 μm²) was used for analysis. Accordingly, a single SMC may have more than one spark site, but the number of wave sites in a vessel corresponds to the number of cells in that vessel producing a wave during the recording period. Because of the much larger size of ROI used to analyze Ca²⁺ waves, sparks occurring within these ROIs are averaged out during the analysis of waves. For both spark and wave analysis, SparkAn measured a running baseline and performed an automatic background subtraction for each event, accounting for variations in background fluorescence between cells, vessels or animals. The resulting data were imported into Microsoft Excel. The “BlueFireGreen” Look Up Table (LUT) was used to pseudocolor images in Image J (NIH).

Diameter measurements of denuded MAs

Following Ca²⁺ imaging, vasomotor responses of denuded arteries were tested in response to NE (1 μM) to ensure SMC viability (constriction to NE), and to ACh (1 μM) during NE preconstruction to ensure disruption of endothelium (lack of dilation). Internal diameter (ID) was measured using edge-tracking software provided by Dr. Michael Davis (University of Missouri) using LabView 2009 (National Instruments, Austin, TX, USA). Diameter images were acquired using a 10X water immersion objective (NA = 0.3) and a Firewire camera (DMK 21AF04; TheImagingSource; Charlotte, NC, USA).

Serum CGRP measurements

Serum samples were prepared by allowing blood collected via cardiac puncture to clot in a 1.5 ml centrifuge tube for 30 min at room temperature. Samples were then centrifuged in a Sorvall Legend Microcentrifuge for 15 min at 1500xG at 4°C. Serum was immediately transferred to a clean 1.5 ml tube and stored at −80°C until use in assays. Thawed samples were analyzed simultaneously in triplicate according to manufacturer’s protocol using the CGRP Enzyme Immunoassay Kit for Rat and Mouse (Phoenix Pharmaceuticals, EK-015–09). Completed assays were read using a microplate reader (SpectraMax M2E; Molecular Devices; San Jose, CA USA).

Smooth muscle cell isolation and immunostaining

Second-order MAs (~6 per mouse) were collected from young and old mice and cut into 1–2 mm segments, which were transferred into 12 × 75 mm culture tubes containing 4 ml of chilled (4°C) dissection buffer containing (in mM): 137 NaCl, 5 KCl, 1 MgCl₂, 10 HEPES, 10 glucose, 0.01 sodium nitroprusside (SNP) and 0.1% BSA. The culture tube was warmed to room temperature, and segments were transferred to a culture tube containing 1 ml of preheated (37°C) dissociation buffer containing (in mM): 137 NaCl, 5 KCl, 1 MgCl₂, 10 HEPES, 10 glucose, 2 CaCl₂, 0.1% BSA, 0.62 mg ml⁻¹ papain, 1.5 mg ml⁻¹ collagenase H and 1.0 mg ml⁻¹ dithioerythritol, pH 7.4. Vessel segments were incubated for 25 min at 37°C then transferred to enzyme-free dissociation buffer at room temperature. Tube volume was reduced to 600 μl and vessel segments were rapidly triturated 3X using a 100 μl Eppendorf-style pipette. 200 μl of the resulting cell suspension was transferred to a microscope slide coated with laminin (Cat. #23017–015; Life Technologies; Carlsbad, CA, USA; 4 μl per slide) and allowed to adhere for 60 min.

Smooth muscle cells adhered to slides were fixed in 4% paraformaldehyde for 15 min, gently washed twice in phosphate-buffered saline (PBS; Cat. #P5368; Sigma), blocked with 10% normal goat serum and permeabilized for 30 min in PBS containing 10% normal goat serum and 0.1% Triton X100, then washed with PBS alone. Thus prepared, slides were incubated 60 min in primary antibody [Alamone Labs, 1:250] for RyR1 (Cat. #ARR-001), RyR2 (Cat. #ARR-002), or RyR3 (Cat. #ARR-003). After washing in PBS, slides were incubated in secondary antibody (goat-anti-rabbit 488; Cat. # A-11008; ThermoFisher) for 60 min, washed a final time in PBS and cover slipped on slides using ProLong Gold mounting media (Life Technologies). During imaging, SMCs were differentiated from ECs based on cell morphology: any cells lacking an elongated, spindle shape were excluded. Control experiments included verifying a lack of fluorescent staining using the above protocol with corresponding blocking peptides together with the primary antibody as well as secondary antibody-only controls. Slides were imaged using conventional fluorescence on a Nikon E800 microscope. Images were acquired using the Extended Depth of Focus feature of Nikon Elements software to create a composite 2D image from a series of manual Z-steps. For all slide sets, slides were imaged using similar exposure and gain settings.

Chemicals and Reagents

All chemicals and reagents were obtained from MilliporeSigma (Burlington, MA, USA) unless otherwise noted. Solutions were prepared fresh daily. Fluo-4 AM was dissolved in DMSO (<0.1% final concentration during incubation). All other drug solutions were prepared in control PSS.

Data presentation and statistical analyses

All summary data are reported as mean ± SEM, with statistical significance determined at P < 0.05. Experimental data were analyzed as follows:

Calcium Events

Data for event sites, frequency, amplitude and full duration half maximum (FDHM) are presented for each vessel under respective conditions, with each condition delineated by a bar depicting mean ± SEM. Event data were analyzed in Graphpad Prism 8 (San Diego, CA, USA) via Two-way ANOVA with repeated measures between pharmacological treatments, followed by Sidak multiple comparison tests.

Vessel Diameters

MA diameters measured following denudation were analyzed by paired t-tests across pharmacological treatments followed by correction for false discovery by Benajamini-Hochberg correction in GraphPad Prism.

RT-qPCR data

As described²², unpaired t-tests were used to compare average C_T values from replicates within each age group. Because the genes measured for this study were included in a larger array of data²², P-values were adjusted to account for multiple comparisons using the Benjamini- Hochberg procedure. Fold-changes in gene expression in samples from old vs. young mice were determined using the 2^−ΔΔCT method³⁸.

Serum CGRP measurements

The mean value of samples run in triplicate were plotted using the standard curve (R² = 0.9996), analyzed using unpaired t-test in GraphPad Prism.

Results

Representative Ca²⁺spark and wave

A representative Ca²⁺ spark and Ca²⁺ wave in adjacent SMCs of a MA are shown in Figure 1. The spatial domain, amplitude and FDHM of Ca²⁺ sparks were each characteristically smaller than for Ca²⁺ waves. For Figures 2 and 3, data for Ca²⁺ sparks and waves are first presented for MAs with intact endothelium and then for MAs with denuded endothelium. Under respective conditions, data are first presented with respect to age followed by addition of NE and then of CGRP.

Figure 1: Representative Ca²⁺ spark and Ca²⁺ wave in Fluo 4-loaded SMCs.

(A). Representative confocal frames showing Fluo 4-fluorescence before (Upper Panel) and during a (Lower Panel) Ca²⁺ spark and Ca²⁺ wave in mouse MA. Arrows mark same location in respective panels. Dotted line depicts inner diameter of cannulated MA. Red boxes in lower panel represent ROIs used to generate representative traces with pseudocolored images shown below. (B-C) Representative traces of the Ca²⁺ spark (B) and Ca²⁺ wave (C) depicted in A.

Figure 2: Properties of Ca²⁺ sparks in SMCs from intact and denuded MAs.

Data are per FOV for: (A) Active spark sites (B) spark Frequency (C) spark Amplitude, and (D) Full duration at half-maximal amplitude (FDHM). Sparks were analyzed with MAs in control PSS, during preconstriction with age-specific EC₅₀ of NE for young (167 nM) and old (600 nM), and during exposure to EC₅₀ NE + CGRP (100 nM). Data are means ± SEM with individual data for each MA tested [n = 14–18 (intact) and 7–8 (denuded) per age group]. # P<0.05 vs. young; * P<0.05 vs. PSS; + P<0.05 vs. NE; ≈ P<0.05 vs. intact. (E) Representative image of ROI placement and (F) intensity (F/F_o) trace of 4 ROIs placed on active spark sites in MA from young mouse in PSS depicting 17 total sparks. Nerves and adventitial cells are apparent along vessel edges.

Figure 3: Properties of Ca²⁺ waves in SMCs from intact and denuded MAs.

Data are per FOV for: (A) Active wave sites (B) wave Frequency (C) wave Amplitude and (D) Full duration at half-maximal amplitude (FDHM). Waves were analyzed with MAs in control PSS, during preconstriction with age-specific EC₅₀ of NE for young (167 nM) and old (600 nM), and during exposure to EC₅₀ NE + CGRP (100 nM). Data are means ± SEM with individual data for n = 14–18 (intact) and 7–8 (denuded) MAs per age group. # P<0.05 vs. young; * P<0.05 vs. PSS; + P<0.05 vs. NE; ≈ P<0.05 vs. intact. (E) Representative image of ROI placement and (F) intensity (F/F_o) trace of 4 ROIs separate SMCs with Ca²⁺ wave sites in an MA from a young mouse in PSS depicting 4 total waves.

Mesenteric arteries with intact endothelium

Aging decreases spontaneous SMC Ca²⁺ spark sites and increases Ca²⁺ wave frequency

When perfused with control PSS, MAs from young mice had 8.5 ± 1.3 spontaneously active spark sites in 10 s per FOV vs. 4.1 ± 0.4 (P < 0.05) active sites in MAs from old mice (Figure 2). There were no significant differences in young vs. old for the frequency (0.063 ± 0.01 vs. 0.077 ± 0.01 events/s), amplitude (1.29 ± 0.01 vs. 1.30 ± 0.01 F/F_o) or FDHM (0.16 ± 0.01 vs. 0.15 ± 0.01 s). Wave sites (i.e., number of SMCs per FOV producing Ca²⁺ waves, 6.6 ± 0.8 vs. 6.9 ± 0.7) and wave amplitudes (2.1 ± 0.1 vs. 1.8 ± 0.1 F/F_o) were similar in MAs from young vs. old mice, while the frequency of waves was greater in MAs from old vs. young mice (0.027 ± 0.003 vs. 0.044 ± 0.003 events/s, P < 0.05) and was accompanied by decreased FDHM vs. MAs from young mice (7.3 ± 0.6 vs. 4.7 ± 0.4 s, P < 0.05, Figure 3). Thus, aging decreases spontaneous Ca²⁺ spark sites without a change in spark kinetics or amplitude. In contrast, Ca²⁺ waves shift towards more frequent events of shorter FDHM with aging.

Norepinephrine-induced SMC Ca²⁺ signals differ for intact MAs from young vs. old mice

Norepinephrine, applied at age-specific EC₅₀ levels to preconstrict intact MAs at similar levels of sympathetic SMC activation, significantly decreased the number of Ca²⁺ spark sites per FOV in MAs from both young (8.6 ± 1.3 to 3.1 ±0.7) and old (4.1 ± 0.4 to 0.8 ± 0.2) mice, with significantly fewer active sites remaining in MAs from old vs. young mice (Figure 2A). These reductions in spark sites occurred without changes in their frequency (Figure 2B), amplitude (Figure 2C) or FDHM (Figure 2D) from either age group.

Concurrent with the decrease in Ca²⁺ spark sites, NE significantly increased the number of sites displaying Ca²⁺ waves for MAs from both young (6.6 ± 0.8 to 10.5 ± 1.0 cells) and old (6.9 ± 0.7 to 14.2 ± 0.8 cells) mice and this effect was significantly greater in MAs from old mice (Figure 3A). Norepinephrine also increased wave frequency in MAs from young (0.027 ± 0.003 to 0.038 ± 0.004 events/s) and old (0.044 ± 0.003 to 0.056 ± 0.003 events/s) mice, with significantly greater frequency in MAs from old mice MAs (Figure 3B). Addition of NE did not alter wave amplitude (Figure 3C) yet decreased wave FDHM in MAs from young (7.3 ± 0.6 to 5.4 ± 0.7 s) but not old mice (Figure 3D), which had shorter FDHM (vs. young) with in control PSS.

CGRP counteracts NE-induced changes in SMC Ca²⁺ signals in MAs of young but not old mice

CGRP (100 nM) was added to MAs that were preconstricted with NE (EC₅₀, as above) to mimic sensory nerve activation during sympathetic neuroeffector signaling. Following NE-induced decreases in Ca²⁺ spark sites, CGRP significantly increased spark sites in MAs from young mice (from 3.1 ± 0.7 to 6.4 ± 0.7), but not in MAs from old mice (Figure 2A). Spark frequency (Figure 2B), amplitude (Figure 2C) and FDHM (Figure 2D) were unaffected by CGRP treatment in either age group. For Ca²⁺ waves, addition of CGRP reduced the number of active wave sites in MAs from both young (10.5 ± 1.0 to 5.1 ± 0.8) and old mice (14.1 ± 0.8 to 9.4 ± 0.06); these were no longer different from active sites during control PSS in MAs from young mice but remained significantly higher than control in MAs from old mice (Figure 3A). Addition of CGRP did not alter wave frequency from NE-induced levels in either age group (Figure 3B), but significantly decreased wave amplitude from control and from NE-induced levels in MAs of both young (from 2.1 ± 0.1 to 1.8 ± 0.1 F/Fo) and old (from 2.0 ± 0.05 to 1.7 ± 0.04) mice (Figure 3C). In the presence of NE, addition of CGRP decreased wave FDHM only in MAs from old mice (from 4.4 ± 0.2 to 3.7 s ± 0.2), and to values below those from young mice under the same experimental conditions (Figure 3D).

Mesenteric arteries with denuded endothelium

Endothelial denudation alters SMC Ca²⁺ signaling in MAs from young mice

To determine the role of the endothelium in mediated SMC Ca²⁺ signals under each experimental condition, experiments were repeated in denuded MAs. Overall, disrupting the endothelium altered Ca²⁺ signals to a greater extent in MAs from young vs. old mice. Thus, in MAs from young mice in PSS, denudation significantly decreased spontaneous Ca²⁺ spark sites (8.6 ± 1.3 to 5.1 ± 0.6, Figure 2A) and increased spark amplitudes (1.3 ± 0.01 to 1.4 ± 0.02 F/F_o, Figure 2C). Spontaneous spark frequency (Figure 2B) and FDHM (Figure 2D) were unchanged by denudation in either age group. During the addition of NE, Ca²⁺ spark sites decreased, spark amplitude increased, while spark frequency and FDHM were unchanged in denuded vs. intact MAs from young and old mice. Whereas NE-decreased Ca²⁺ spark sites were restored by the addition of CGRP in intact MAs of young mice, this did not occur in denuded MAs. In the presence of NE, addition of CGRP increased the number of spark sites in denuded MAs (from 1.85 ± 0.6 to 2.86 ± 0.6) but had no effect on spark frequency, amplitude or FDHM in MAs from young or old mice (Figures 2B–D).

As shown in Figure 3, denuding the endothelium in PSS did not alter the number of active sites for Ca²⁺ waves or their amplitudes in MAs from young mice, yet significantly increased wave frequency (0.028 ± 0.003 to 0.047 ± 0.007 events/s) and decreased FDHM (7.3 ± 0.6 to 4.6 ± 0.5 s). In denuded MAs from young mice, NE increased Ca²⁺ wave sites (7.8 ± 1.2 to 15.4 ± 1.1) and frequency (0.047 ± 0.007 to 0.056 ± 0.006 events/s) vs. respective intact MAs. In MAs from old mice, denudation did not alter NE-induced wave sites, frequency or FDHM but increased amplitude vs. respective intact MAs (1.9 ± 0.1 to 2.2 ± 0.2 F/F_o). In denuded MAs from young mice, addition of CGRP significantly decreased active wave sites vs. NE alone (15.4 ± 1.1 to 11.0 ± 1.5), but these remained significantly higher than the active sites in respective intact MAs (5.1 ± 0.8); neither wave frequency, amplitude nor FDHM differed from those of respective intact MAs (Figures 3B–D). In MAs from old mice, denudation did not alter CGRP-mediated effects on the number of active wave sites, frequency or FDHM compared to intact MAs but increased wave amplitude vs. intact (1.7 ± 0.04 to 2.0 ± 0.1 F/Fo) (Figure 3C).

Pharmacological validation of Ca²⁺ sparks

To validate Ca²⁺ sparks using established pharmacological agents⁵ Ca²⁺ sparks in SMCs of MAs from young and old mice were evaluated in the presence and absence of the RyR agonist caffeine (1 mM) and the RyR antagonist ryanodine (10 μM). MAs from young mice exhibited significantly more spontaneous spark sites vs. MAs from old mice (8.2 ± 0.4 vs. 4.4 ± 0.3) (Figure 4), consistent with independent data from separate mice in Figure 2. Caffeine increased spark sites in MAs of both young and old mice (to 13.5 ± 0.6 and 9.5 ± 0.6, respectively) compared to PSS, with the number of sites per FOV remaining significantly higher in MAs from young vs. old mice. Caffeine did not alter spark frequency, amplitude or FDHM in MAs from young or old mice. Addition of ryanodine eliminated nearly all active sites for sparks in both age groups (Figure 3A), and significantly decreased the frequency (young: 0.04 ± 0.008 events/s; old: 0.04 ± 0.012 events/s) of remaining sparks (Figure 4B), with no change in amplitude or FDHM (Figure 4C–D).

Figure 4: Pharmacological validation of Ca²⁺ sparks.

Sparks were analyzed in MAs from young and old mice with vessels immersed in PSS (Control), during exposure to caffeine (1 mM) and to ryanodine (10 μM). Data are means ± SEM with individual vessel data for (A) Spark (B) Frequency (C) Amplitude and (D) FDHM. n = 14–18 (intact) and 7–8 (denuded) MAs per group. # P<0.05 vs. young; * P<0.05 vs. PSS; + P<0.05 vs. caffeine.

Confirmation of endothelial denudation

Following Ca²⁺ measurements, denuded arteries were washed in control PSS and exposed to EC₅₀ of NE, to NE + ACh (1 μM), and to 0 Ca²⁺ PSS; vessel ID was recorded under each condition (Figure 5). Denuded MAs from both young and old mice constricted similarly to respective NE stimuli, in accord with previous studies³⁵. Acetylcholine failed to dilate preconstricted MAs (confirming EC disruption in all but 2 vessels, which were excluded from the study), while incubation in 0 Ca²⁺ PSS increased diameters slightly but significantly above those recorded in control PSS. Any vessels lacking ≥ 50% constriction to 1 μM NE (n=2) or that dilated to 1 μM ACh following endothelial denudation (n=0) were excluded from the study. The integrity of vasoconstriction to NE and lack of vasodilation to ACh confirm SMC viability and effective endothelial disruption with our denudation procedure.

Figure 5: Vasomotor responses following endothelial denudation.

Data are means ± SEM with individual data for internal diameter of MAs from young and old mice. Following denudation, all MAs had similar resting control (PSS) and maximum (0 Ca²⁺) diameters, constrictions to EC₅₀ of NE (young: 167 nM, old: 600 nM), and lack of vasodilation to ACh (1 μM). n=7–8 MAs per age group. * P < 0.05 vs. PSS, # = P < 0.05 vs. NE, + = P < 0.05 vs. NA + ACh.

Transcript levels of CGRP receptor genes in SMCs and ECs are maintained with aging

Expression of genes for two CGRP receptor proteins, RAMP1 (Ramp1) and CRLR (Calcrl) were measured using quantitative real-time PCR (qPCR) in SMCs and ECs freshly isolated from MAs of young and old mice, where age-related changes in RAMP1 protein localization were previously demonstrated ³⁵. These qPCR data were published within a supplement for a larger analysis²², were re-analyzed for the present study, and presented in Table 1. In both SMCs (Table 1A) and ECs (Table 1B), Ramp1 and Calcrl transcripts were expressed at similar levels. Application of the 2^−ΔΔCT method^38,39, with 18s as the control gene, showed only small differences in expression for SMCs of MAs from old vs. young mice. In SMCs, fold-changes were 3.0 (Ramp1) and 0.7 (Calcrl) (Table 1A). Fold-changes in expression for ECs were 0.4 (Ramp1) and 2.5 (Calcrl) (Table 1B).

Table 1A. Transcript levels for CGRP receptor component genes in mesenteric artery smooth muscle cells from young and old mice.

Data are from²² with permission.

	Young (SMC)			Old (SMC)
	RAMP1	CALCRL	18S	RAMP1	CALCRL	18S
CT	22.2 ± 1.5	25.6 ± 0.6	19.1 ± 0.6	22.08 ± 0.7	26.3 ± 0.9	19.2 ± 0.7
ΔCT	3.1	6.5	0.0	1.5	7.0	0.0
ΔΔCT	--	--	--	−1.5	0.48	0.0
2−ΔΔCT	--	--	--	3.0	0.7	1.0

CT: Average expression cycle threshold (of 40 total cycles)

ΔCT = AvgCT_Target − AvgCT_18S

ΔΔCT = ΔCT_old − ΔCT_Young

2^−ΔΔCT = Fold change in expression vs. young

Table 1B. Transcript levels for CGRP receptor component genes in mesenteric artery endothelial cells from young and old mice.

Data are from²² with permission.

	Young (EC)			Old (EC)
	RAMP1	CALCRL	18S	RAMP1	CALCRL	18S
CT	26.2 ± 1.1	22.9 ± 2.0	18.1 ± 0.6	27.3 ± 0.7	21.2 ± 0.9	17.7 ± 0.7
ΔCT	8.1	4.8	0.0	9.6	3.5	0.0
ΔΔCT	--	--	--	1.5	−1.3	0.0
2−ΔΔCT	--	--	--	0.4	2.5	1.0

Definitions as in Table 1A

Aging alters RyR isoform transcript and protein expression in SMCs

To determine whether altered RyR isoform expression contributes to age-related changes in Ca²⁺ signaling, we measured the expression of Ryr1, Ryr2 and Ryr3 genes in SMCs using qPCR. These data were originally published with the above²² and are presented in Table 1C. In SMCs of MAs from young mice, Ryr2 was the most highly-expressed isoform (according to C_T values), with less expression of Ryr1 and no detectable expression of Ryr3 (Table 1C). In SMCs from old mice, Ryr2 expression remained highest, Ryr3 was expressed, and Ryr1 was no longer detected. Fold-changes in expression of each isoform in SMCs from old vs. young mice were 0.003 (Ryr1), 3.8 (Ryr2) and 1925 (Ryr3) (Table 1C). Endothelial cells are reported to not express functional RyRs^40,41.

Table 1C. Transcript levels for ryanodine receptor isoforms in mesenteric artery smooth muscle cells from young and old mice.

Data are from²² with permission.

	Young				Old
	RyR1	RyR2	RyR3	18S	RyR1	RyR2	RyR3	18S
CT	30.9 ± 0.2	21.5 ± 1.9	40.0 ± 0.0	17.5 ± 0.7	40.0 ± 0.2*	20.2 ± 1.9	29.6 ± 0.0*	18.0 ± 0.7
ΔCT	13.4	4.0	22.5	0.0	22.0	2.1	11.6	0.0
ΔΔCT	--	--	--	--	8.6	−1.9	−10.9	0.0
2−ΔΔCT	--	--	--	--	0.003	3.8	1925.2	1.0

Definitions as in Table 1A

Protein expression for each RyR isoform was evaluated in freshly-isolated, fixed SMCs using antibodies for RyR1, RyR2 and RyR3 followed by conventional fluorescence imaging. Expression patterns mirrored those measured using qPCR: images of SMCs from MAs of young mice showed expression of RyR2>RyR1>>RyR3 while those from old mice expressed RyR2>RyR3>>RyR1 (Figure 6).

Figure 6. Aging alters expression of ryanodine receptors in smooth muscle cells.

Representative immunofluorescence images depicting green fluorescence for RyR1 (top rows), RyR2 (center rows) and RyR3 (bottom rows) in 3 separate SMCs from MAs of (A) young and (B) old mice, (C) SMCs incubated with respective blocking peptides, and (D) SMC with primary omitted. ToPro nuclear stain (blue) is included in all images. Scale bars = 20 μm and apply to all panels.

Aging increases serum [CGRP]

Serum CGRP levels in young and old mice were measured by ELISA, in triplicate, to determine whether circulating [CGRP] varied between the age groups studied here. As shown in Figure 7, serum [CGRP] was significantly higher in old (64.4 ± 5.3 pM) vs. young (46.8 ± 5.9 pM) mice.

Figure 7. Advanced age increases serum CGRP.

Data are means ± SEM with individual data for serum concentrations (pM) of CGRP in young and old mice measured by ELISA with interpolation from a standard curve (10 pg ml⁻¹ - 1000 ng ml⁻¹, R² = 0.9996). n = 10 mice per group, * P<0.05 vs. young.

DISCUSSION

This is the first study to investigate how advanced age affects SMC Ca²⁺ signaling in resistance arteries in light of impaired sympathetic vasoconstriction and sensory vasodilation^34,35. In cannulated pressurized MAs of young (3–6 mo) and old (24–26 mo) male C57BL6/J mice, we applied NE to simulate sympathetic nerve activation and CGRP to simulate sensory nerve activation. Our findings show that: 1) The number of spontaneously active Ca²⁺ spark sites (which promote vasodilation via BK_Ca channel activation) decreased with aging, coincident with an increase in the frequency of Ca²⁺ waves that promote vasoconstriction (via crossbridge activation); 2) NE decreased spark sites and increased wave sites and frequency to a greater extent in MA SMCs from old vs. young mice; 3) addition of CGRP during NE stimulation restored SMC Ca²⁺ signaling to control levels in MAs from young (but not old) mice by increasing spark sites and decreasing wave sites; 4) denuding the endothelium disrupted Ca²⁺ spark and wave activity in MAs from young but not old mice, for which Ca²⁺ signals were already depressed; and 5) aging led to disappearance of spark-promoting RyR1 and appearance of the spark-inhibitory RyR3 gene and protein expression in SMCs without affecting the expression of CGRP receptor components. Taken together, these findings implicate altered RyR isoform expression and endothelial dysfunction as key factors contributing impaired basal and neurotransmitter-induced Ca²⁺ signaling in MAs with advanced age and that dysfunctional SMC Ca²⁺ signaling impairs vasodilation to CGRP with aging.

Altered RyR expression inhibits Ca²⁺ sparks with aging

This study is the first to directly evaluate Ca²⁺ sparks in SMCs of resistance arteries during aging. Despite unchanged spark frequency across ages and treatments (Figure 2B), our finding of decreased active spark sites with aging in the mouse (Figure 2A) is consistent with decreased BK_Ca-derived STOCs in SMCs of MAs from older humans (> 65 yr), with concomitant reductions in gene and protein expression for BK_Ca⁴². Decreased spark sites within a given field has a functionally similar effect to decreased frequency, as fewer active sites will lead to a decrease in STOCs when both groups produce sparks at equal frequency. In contrast, transcript levels for BK_Ca were not different between SMCs of MAs from young and old mice corresponding to the age groups studied here²². The relationship between aging, BK_Ca expression and STOC production appears to be vascular bed-specific: STOC frequency increased with aging (~ 4 mo vs. ~24 mo) in the superior epigastric arteries of mice in concert with expression of the pore-forming α-subunit of BK_Ca channels⁴³ while BK_Ca expression and function in rat cerebral arteries was preserved at 25–30 mo vs. 3–5 mo of age⁴⁴.

In light of decreased Ca²⁺ spark sites with aging, we suggest that changes in RyR isoform expression, which varies widely across species and vascular beds⁵, may explain both the decrease in number of SMC sparks sites observed here with aging (Figure 2A) and the variability in findings for age-related STOC frequency across previous studies. That RyR2 is the predominantly-expressed isoform in young MA SMCs (Table 1C and Figure 6) is consistent with findings in isolated SMCs from MAs of adult mice, where expression of RyR2 transcript dominated RyR1 and RyR3 was absent⁴⁵. In cerebral arteries of rats, expression of RyR2 protein also dominated RyR1 and RyR3⁴⁶. Our finding of increased RyR3 and decreased RyR1 gene and protein expression with fewer spark sites is consistent with previous studies demonstrating²¹ or inferring⁷ an inhibitory role for RyR3 (or a spice variant thereof⁴⁷) on Ca²⁺ sparks in vascular SMCs. Changes in RyR transcript levels (Table 1C) that are consistent with differences in immunostaining for protein (Figure 6), supports this interpretation. Altered RyR isoform expression may also limit the maximal spark-generating capacity of SMCs in MAs of old mice, as caffeine failed to increase the number of active spark sites to the same extent as observed for young mice (Figure 4A). Supporting this interpretation are studies showing that RyR3 does not participate in Ca²⁺ spark generation^20,48. Further, the amplitude of caffeine-induced sparks was lower in SMCs of old vs. young mice (Figure 4C). While Ca²⁺ store content was not evaluated here, it is maintained in SMCs of human MAs⁴² and may increase in SMCs of mouse MAs⁴⁹ with aging. Collectively, these findings support the conclusion that altered RyR isoform expression underlies decreases in spontaneous Ca²⁺ spark sites in SMCs from mouse MAs during aging.

Aging alters NE-induced Ca²⁺ waves properties but not vasoconstriction

Changes in Ca²⁺ sensitization with aging are suggested by increased frequency (Figure 3B) with decreased FDHM (Figure 3D) of Ca²⁺ waves. Aging is associated with NE-desensitization throughout the body, thus we controlled for altered adrenergic sensitivity by using age-specific EC₅₀ of NE³⁴ and investigated Ca²⁺ signals at similar levels of sympathetic (adrenergic) vasoconstriction. Aging was also associated with proportionally greater increases in NE-induced Ca²⁺ waves sites (Figure 3A) and wave frequency (Figure 3B). Thus, SMCs in aged MAs produce more Ca²⁺ waves to generate equivalent levels of vasoconstriction, consistent with studies of Fura-2 Ca²⁺ and diameter measurements in NE-treated MAs of aged (70–100 wk) rats⁵⁰. In contrast, isolated MA SMCs showed age-related decreases in whole-cell Fura-2 Ca²⁺ signals when exposed to the α₁-selective adrenoreceptor agonist phenylephrine⁵¹, suggesting potential roles for the α₁- and/or β-adrenergic signaling pathways, which are also activated by NE. The changes observed here for Ca²⁺ waves in concert with reduced sensitivity to NE³⁴ are consistent with age-related Ca²⁺ desensitization in response to increased sympathetic drive with advancing age in humans⁵².

Aging impairs CGRP inhibition of sympathetic SMC activation via altered Ca²⁺ signals

A primary goal of this study was to investigate the influence of the sensory neurotransmitter CGRP on SMC Ca²⁺ signaling during sympathetic activation with NE. Following preconstriction with age-specific EC₅₀ levels of NE, vasodilation to CGRP is impaired in MAs of old vs. young mice, particularly at 10–100 nM³⁵. Thus, from similar (EC₅₀) levels of sympathetic activation, we applied 100 nM CGRP to investigate its effect on Ca²⁺ signals. CGRP restored the number and amplitude of Ca²⁺ waves (Figures 3A and 3C), with a concurrent increase in the number of Ca²⁺ spark sites (Figure 2A) in MAs of young but not old mice. Both events were restored to control (pre-NE) numbers of active sites following the addition of CGRP only in young mice, suggesting that sensory inhibition of sympathetic vasoconstriction is impaired with aging. In the presence of equal sympathetic activation, these findings imply that dysfunctional CGRP-mediated Ca²⁺ signaling contributes to impaired MA dilation with aging.

The reduction in CGRP-mediated Ca²⁺ sparks with aging implicates a role for RyRs in decreased CGRP sensitivity and vasodilation. The replacement of spark-generating RyR1 expression with spark-decreasing RyR3 expression (Table 1C, Figure 6) supports a role for RyR isoform switching in the impairment of CGRP-mediated sympathetic inhibition because aging decreases the ability of CGRP to produce enough Ca²⁺ sparks to blunt NE-induced Ca²⁺ waves and vasoconstriction. However, decreased spark activity may also result from CGRP-induced, protein kinase A- and/or protein kinase C-mediated phosphorylation of RyRs, as CGRP receptor signaling includes both PKA and (to a lesser extent) PKC activation^53,54. Such an effect is consistent with findings that protein kinase C-mediated phosphorylation of RyRs decreases sparks in cerebral artery SMCs^55,56. Age-related changes in Ca²⁺ signaling could also result from CGRP-independent changes to RyRs, such as oxidation, as RyRs are redox-sensitive⁵⁷ and vascular levels of reactive oxygen species increase with advancing age^25,58. Thus, spontaneous and CGRP-mediated Ca²⁺ signals in SMCs are effected by RyR function through changes in isoform expression and by modulating their activity. Additional studies are needed to more-precisely define the role of RyR isoform expression vs. posttranslational modifications underlying the age-related decline in CGRP modulation of Ca²⁺ sparks.

Endothelial denudation in young MAs mimics the effect of aging on Ca²⁺ signaling

The endothelium contributes to CGRP-mediated dilation of MAs³⁵ but vasodilation to CGRP in other vascular beds is endothelium-independent^59,60. Under all conditions of the present study, denudation significantly altered Ca²⁺ sparks and waves in MAs from young, but not old, mice (Figures 2 and 3). Functionally, removal of the endothelium caused SMCs of young MAs to behave like those of old MAs as shown by: 1) decreased basal Ca²⁺ sparks sites; 2) failure of CGRP to increase sparks during NE preconstriction; 3) increased NE-induced Ca²⁺ waves; and 4) failure of CGRP to decrease Ca²⁺ waves to the extent observed in intact MAs. The pronounced reduction in spontaneous Ca²⁺ sparks following denudation of MAs from young mice (Figure 2A) is consistent with such an effect in cerebral arteries⁶¹, where subsequent addition of an NO donor restored spark activity. These findings suggest that, during aging, loss of endothelial function contributes to sensory nerve dysfunction in regulating SMC Ca²⁺ signaling and vasomotor control. In turn, we suggest that sensory neurotransmission can promote vasodilation through myoendothelial communication.

The role of the endothelium and specific signaling mechanisms underlying CGRP vasodilation in MAs of both young and aged mice remains unclear. Previous studies highlight endothelial participation in CGRP dilation of MAs exclusively from old mice³⁵, where there is altered localization of the CGRP receptor protein, RAMP1, in ECs³⁵ and impaired CGRP-mediated SMC Ca²⁺ signaling is evident (Figures 2–3). Expression of CGRP receptor genes Ramp1 and Calcrl in freshly isolated SMCs (Table 1A) and ECs (Table 1B) were similar with aging, thus they do not explain differences in Ca²⁺ signaling observed between age groups in this study. However, increased circulating (serum) CGRP levels with aging (Figure 7) may contribute to changes in receptor function in the absence of altered gene expression because chronic exposure to CGRP leads to receptor desensitization, internalization and degradation⁵³. Supporting a non-CGRP receptor-mediated pathway for CGRP is a recent study in rat MAs⁶², which found that CGRP released from sensory nerves inhibited endothelial-mediated vasodilation via pannexin channel opening leading to the inhibition of NO production. In light of elevated serum CGRP with aging (Figure 7), this signaling pathway may contribute to the decreased role of the endothelium in affecting SMC Ca²⁺ signaling with advanced age. Future studies will need to account for the role of NO production vs. sensory nerve-derived CGRP on endothelial autacoid production and vasomotor function. Resolving the effects of a NO synthase inhibitor on downstream signaling events should provide additional insight.

Summary and Conclusion

The goal of this study was to identify the effects of aging on SMC Ca²⁺ signals (sparks and waves) underlying impaired sympathetic vasoconstriction and sensory vasodilation in mouse MAs. Exposure of isolated pressurized MAs to functionally-defined concentrations of NE and CGRP (and thereby mimic the actions of respective neurotransmitters) revealed significant changes in Ca²⁺ signaling with aging. The SMCs of MAs from old mice exhibited enhanced NE-induced Ca²⁺ waves that were not diminished by exposure to CGRP. These SMCs exhibited few spontaneously active Ca²⁺ spark sites and, unlike young SMCs, did not increase the number of active spark sites when exposed to CGRP. Collectively, the increase in Ca²⁺ waves with reduced spark activity would promote vasoconstriction with aging, as would the increased expression of spark-inhibiting RyR3. Endothelial denudation increased NE-induced waves and decreased spontaneous and CGRP-induced sparks in young MAs but had little effect on Ca²⁺ signals in old MAs. Our findings suggest that alterations in spontaneous and neurotransmitter-mediated SMC Ca²⁺ signaling contribute to vascular dysfunction during advancing age with a diminished role for the endothelium in modulating the actions of CGRP.

Perspective.

Advanced age is the greatest risk factor for cardiovascular disease. Vascular dysfunction during aging is associated with increased sympathetic drive and impaired ability to overcome sympathetic vasoconstriction. However, little is known of how advanced age affects perivascular innervation of resistance vessels controlling tissue blood flow. Arteries supplying the mesenteric circulation are dually innervated by sympathetic (i.e., vasoconstrictor) and sensory (i.e., vasodilator) nerves. We have shown that advanced age reduces the ability of perivascular sensory nerves to inhibit sympathetic constriction and dilate mesenteric arteries^34,35. In the present study, we investigated how neurotransmitter-evoked Ca²⁺ signaling in smooth muscle cells of mesenteric arteries is affected by advanced age. Our findings reveal that, with aging, there is an imbalance between the global intracellular Ca²⁺ waves that produce vasoconstriction and the localized Ca²⁺ sparks that mediate vasodilation. This imbalance is manifest under resting conditions, accentuated during stimulation with the sympathetic neurotransmitter, norepinephrine, and persists during concomitant stimulation with the sensory neurotransmitter, calcitonin gene related peptide. These changes in intracellular Ca²⁺ signaling during advanced age promote vasoconstriction, coincide with altered ryanodine receptor isoform expression and endothelial dysfunction. A novel strategy for improving vascular function centers on preserving the ability of perivascular sensory nerves to oppose sympathetic vasoconstriction and thereby promote tissue perfusion, organ function and preserve the quality of life with advanced age.

Abbreviations

BK_Ca

large conductance calcium-activated potassium channels

[Ca²⁺]_i

intracellular Ca²⁺ concentration

CGRP

calcitonin gene-related peptide

CICR

calcium-induced calcium release

DMSO

dimethyl sulfoxide

EC

endothelial cell

FDHM

full duration half maximum

ID

Internal diameter

IP₃

inositol-1,4,5-trisphosphate

LUT

look up table

NE

norepinephrine

NO

nitric oxide

OD

Outer diameter

qPCR

quantitative real-time PCR

RAMP1

receptor-activated modifying protein 1

RyR

ryanodine receptor

SMC

smooth muscle cell

STOC

spontaneous transient outward current

SR

sarcoplasmic reticulum

VGCC

voltage gated calcium channel