The roles played by ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP₃Rs) in vascular smooth muscle in the microcirculation remain unclear. Therefore, the function of both RyRs and IP₃Rs in Ca(²+) signals and myogenic tone in hamster cremaster muscle feed arteries and downstream arterioles were assessed using confocal imaging and pressure myography. Feed artery vascular smooth muscle displayed Ca(²+) sparks and Ca(²+) waves, which were inhibited by the RyR antagonists ryanodine (10 μM) or tetracaine (100 μM). Despite the inhibition of sparks and waves, ryanodine or tetracaine increased global intracellular Ca(²+) and constricted the arteries. The blockade of IP₃Rs with xestospongin D (5 μM) or 2-aminoethoxydiphenyl borate (100 μM) or the inhibition of phospholipase C using U-73122 (10 μM) also attenuated Ca(2+) waves without affecting Ca(²+) sparks. Importantly, the IP₃Rs and phospholipase C antagonists decreased global intracellular Ca(2+) and dilated the arteries. In contrast, cremaster arterioles displayed only Ca(²+) waves: Ca(²+) sparks were not observed, and neither ryanodine (10-50 μM) nor tetracaine (100 μM) affected either Ca(²+) signals or arteriolar tone despite the presence of functional RyRs as assessed by responses to the RyR agonist caffeine (10 mM). As in feed arteries, arteriolar Ca(²+) waves were attenuated by xestospongin D (5 μM), 2-aminoethoxydiphenyl borate (100 μM), and U-73122 (10 μM), accompanied by decreased global intracellular Ca(²+) and vasodilation. These findings highlight the contrasting roles played by RyRs and IP₃Rs in Ca(²+) signals and myogenic tone in feed arteries and demonstrate important differences in the function of RyRs between feed arteries and downstream arterioles.

Key points• Feed arteries and arterioles, respectively, control the magnitude and distribution of blood flow to skeletal muscle but regional differences in the regulation of vasomotor tone are poorly understood.• To provide this insight, we investigated functional roles and molecular expression of the calcium-release channels, ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP 3 Rs) in smooth muscle cells (SMCs) of isolated pressurized vessels of mice.• In feed arteries, SMCs displayed localized calcium sparks and more global calcium waves. In arterioles, SMCs exhibited only calcium waves.• Calcium signalling and vasomotor tone were governed by both RyRs and IP 3 Rs in feed arteries, while only IP 3 Rs were functional in arterioles. Regional differences were also manifest in the expression profile of RyR isoforms.• This new perspective offers the potential for developing novel strategies to target therapeutic interventions to selective regions of vascular beds.Abstract We tested the hypothesis that vasomotor control is differentially regulated between feed arteries and downstream arterioles from the cremaster muscle of C57BL/6 mice.

Objective Intercellular conduction of electrical signals underlies spreading vasodilation of resistance arteries. Small and intermediate-conductance Ca2+ activated K+ channels (SKCa/IKCa) of endothelial cells serve a dual function by initiating hyperpolarization and modulating electrical conduction. We tested the hypothesis that the regulation of electrical signaling by SKCa/IKCa is altered with advancing age. Approach and Results Intact endothelial tubes (60 μm wide; 1-3 mm long) were freshly isolated from male C57BL/6 mouse (Young: 4-6 months; Intermediate: 12-14 months; Old: 24-26 months) superior epigastric arteries. Using dual intracellular microelectrodes, current was injected (±0.1-3 nA) at site 1 while recording membrane potential (Vm) at site 2 (separation distance: 50-2000 μm). Across age groups, greatest differences were observed between Young and Old. Resting Vm in Old (−38±1 mV) was more negative (P<0.05) than Young (−30±1 mV). Maximal hyperpolarization to both direct (NS309) and indirect (acetylcholine) activation of SKCa/IKCa was sustained (ΔVm ~ −40 mV) with age. The length constant (λ) for electrical conduction was reduced (P<0.05) from 1630±80 µm (Young) to 1320±80 μm (Old). Inhibiting SKCa/IKCa with apamin + charybdotoxin or scavenging H2O2 with catalase improved electrical conduction (P<0.05) in Old. Exogenous H2O2 (200 μM) in Young evoked hyperpolarization and impaired electrical conduction; these effects were blocked by apamin + charybdotoxin. Conclusions Enhanced current loss through KCa activation impairs electrical conduction along the endothelium of resistance arteries with aging. Attenuating the spatial domain of electrical signaling will restrict the spread of vasodilation and thereby contribute to blood flow limitations associated with advanced age.

The control of vascular resistance and tissue perfusion reflect coordinated changes in the diameter of feed arteries and the arteriolar networks they supply. Against a background of myogenic tone and metabolic demand, vasoactive signals originating from perivascular sympathetic and sensory nerves are integrated with endothelium-derived signals to produce vasodilation or vasoconstriction. PVNs release adrenergic, cholinergic, peptidergic, purinergic, and nitrergic neurotransmitters that lead to SMC contraction or relaxation via their actions on SMCs, ECs, or other PVNs. ECs release autacoids that can have opposing actions on SMCs. Respective cell layers are connected directly to each other through GJs at discrete sites via MEJs projecting through holes in the IEL. Whereas studies of intercellular communication in the vascular wall have centered on endothelium-derived signals that govern SMC relaxation, attention has increasingly focused on signaling from SMCs to ECs. Thus, via MEJs, neurotransmission from PVNs can evoke distinct responses from ECs subsequent to acting on SMCs. To integrate this emerging area of investigation in light of vasomotor control, the present review synthesizes current understanding of signaling events that originate within SMCs in response to perivascular neurotransmission in light of EC feedback. Though often ignored in studies of the resistance vasculature, PVNs are integral to blood flow control and can provide a physiological stimulus for myoendothelial communication. Greater understanding of these underlying signaling events and how they may be affected by aging and disease will provide new approaches for selective therapeutic interventions.

Key points• Neural control of the circulation is integral to the regulation of tissue blood flow and systemic blood pressure. Vascular dysfunction occurs with ageing but little is known of corresponding changes in the role(s) of perivascular nerves.• We developed a preparation to study intact mesenteric arteries (MAs) in anaesthetized mice to investigate age-related changes in the function of perivascular sympathetic and sensory nerves in vivo.• Ageing decreased the diameter of MAs, reduced their sensitivity to α 1 -adrenoreceptor stimulation and impaired the ability of sensory nerves to attenuate sympathetic vasoconstriction.• These changes were manifest in males and females and were unaffected by the expression of the GCaMP2 transgene in endothelial cells, confirming the utility of this model.• Our results imply that ageing imposes structural and functional limitations to the splanchnic circulation that impair the ability to mobilize blood from the gut in times of physical stress.Abstract Mesenteric arteries (MAs) are studied widely in vitro but little is known of their reactivity in vivo. Transgenic animals have enabled Ca 2+ signalling to be studied in isolated MAs but the reactivity of these vessels in vivo is undefined. We tested the hypothesis that ageing alters MA reactivity to perivascular nerve stimulation (PNS) and adrenoreceptor (AR) activation during blood flow control. First-(1A), second-(2A) and third-order (3A) MAs of pentobarbital-anaesthetized Young (3-6 months) and Old (24-26 months) male and female Cx40 BAC -GCaMP2 transgenic mice (C57BL/6 background; positive or negative for the GCaMP2 transgene) were studied with intravital microscopy. A segment of jejunum was exteriorized and an MA network was superfused with physiological salt solution (pH 7.4, 37• C). Resting tone was ≤ 10% in MAs of Young and Old mice; diameters were ∼5% (1A), 20% (2A) and 40% (3A) smaller (P ≤ 0.05) in Old mice. Throughout MA networks, vasoconstriction increased with PNS frequency (1-16 Hz) but was ∼20% less in Young vs. Old mice (P ≤ 0.05) and was inhibited by tetrodotoxin (1 μM). Capsaicin (10 μM; to inhibit sensory nerves) enhanced MA constriction to PNS (P ≤ 0.05) by ∼20% in Young but not Old mice. Phenylephrine (an α 1 AR agonist) potency was greater in Young mice (P ≤ 0.05) with similar efficacy (∼60% constriction) across ages and MA branches. Constrictions to UK14304 (an α 2 AR agonist) were less (∼20%; P ≤ 0.05) and were unaffected by ageing. Irrespective of sex or transgene expression, ageing consistently reduced the sensitivity of MAs to α 1 AR vasoconstriction while blunting the attenuation of sympathetic vasoconstriction by sensory nerves. These findings imply substantive alterations in splanchnic blood flow control with ageing.

We determined the possible role of large-conductance Ca2+-activated K+ (BK) channels in regulation of venous tone in small capacitance veins and blood pressure. In rat mesenteric venous smooth muscle cells (MV SMC), BK channel α- and β1-subunits were co-expressed, unitary BK currents were detected, and single channel currents were sensitive to voltage and [Ca2+]i. Rat MV SMCs displayed Ca2+ sparks and iberiotoxin (IBTX)-sensitive spontaneous transient outward currents (STOCs). Under resting conditions in vitro, rat MV exhibited nifedipine-sensitive spontaneous oscillatory constrictions. Blockade of BK channels by paxilline and Ca2+ sparks by ryanodine constricted rat MV. Nifedipine caused venodilation and blocked paxilline-, KCl (20 mM) and BayK 8644-induced contraction. Acute inhibition of BK channels with IBTX in vivo increased blood pressure and reduced venous capacitance, measured as an increase in mean circulatory filling pressure in conscious rats. BK channel α-subunits and L-type Ca2+ channel α1-C subunits are expressed in murine MV. However, these channels are not functional as murine MV lacked nifedipine-sensitive basal tone and rhythmic constrictions. Murine MV were also insensitive to paxilline, ryanodine, KCl and BayK8644, consistent with our previous studies showing that murine MV do not have BK β1-subunits. These data show that not only there are species-dependent properties in ion channel control of venomotor tone, but also that BK channels are required for rhythmic oscillations in venous tone.

Smooth muscle cells (SMCs) in arterioles from striated muscle display IP 3 receptor-dependent Ca 2+ waves that contribute to global myoplasmic Ca 2+ concentration and myogenic tone. However, the contribution of voltage-gated Ca 2+ channels (VGCC) to these arteriolar Ca 2+ signals is unknown. We tested the hypothesis that Ca 2+ waves depend on Ca 2+ influx through VGCC in cremaster muscle arterioles loaded with Fluo-4 and imaged by confocal microscopy. At rest, with vessels pressurized to 80 cm H 2 O in 2 mM Ca 2+ , arteriolar diameter was 28 ± 2 μm (n = 5), and SMCs displayed Ca 2+ waves with frequency (FREQ) = 0.21 ± 0.06 Hz, occurrence (OCC) = 3.5 ± 1.0 waves/SMC and amplitude (AMP) = 1.7 ± 0.1 F/Fo. Removal of extracellular Ca 2+ dilated the arterioles to 39 ± 1 μm, and inhibited Ca 2+ waves (FREQ = 0.1 ± 0.03, OCC = 1.7 ± 0.5 waves/SMC and AMP = 1.4 ± 0.06 F/Fo; p < 0.05 vs. rest) indicating that Ca 2+ waves depended, in part, on influx of extracellular Ca 2+ . Similarly, the VGCC antagonist, nifedipine (1 μM), dilated the arterioles to 34 ± 1.3 μm and also inhibited Ca 2+ waves (FREQ = 0.07 ± 0.02 Hz, OCC = 1.1 ± 0.5 waves/SMC, AMP = 1.4 ± 0.05 F/Fo; p < 0.05 vs. rest). Hyperpolarization of SMCs with the K + channel agonist, cromakalim (10 μM), dilated arterioles from 49 ± 3 to 59 ± 4 μm (n = 4, p < 0.05) and also reduced Ca 2+ wave FREQ (0.1 ± 0.04 to 0.03 ± 0.003 Hz), OCC (1.7 ± 0.04 to 0.5 ± 0.05 waves/SMC) and AMP (1.5 ± 0.04 to 1.2 ± 0.004 F/Fo) (p < 0.05). Conversely, depolarization of SMCs with the BK Ca channel blocker, TEA (1 mM), constricted arterioles from 28 ± 2 to 16 ± 1 μm (n = 5, p < 0.05) and increased wave FREQ (0.2 ± 0.1 to 0.5 ± 0.1 Hz, p < 0.05) and OCC (4 ± 1 to 8 ± 2 waves/SMC, p < 0.05), effects blocked by nifedipine (1μM) (p < 0.05). Similarly, in arterioles pressurized to 20 cm H 2 O to eliminate myogenic tone and reduce basal VGCC activity, application of the VGCC agonist, BayK 8644 (5 nM) constricted the arterioles from 14 ± 1 to 8 ± 1 μm and increased wave FREQ (0.2 ± 0.1 to 0.6 ± 0.1 Hz) and OCC (3 ± 1 to 10 ± 1 waves/SMC) (n = 6; p < 0.05), effects that were independent of ryanodine receptors, as Ca 2+ waves were unaffected by ryanodine (50 μM) in the absence or presence of BayK 8644 (n = 6; p > 0.05). These data support the hypothesis that Ca 2+ waves in arteriolar SMCs depend, in part, on Ca 2+ influx through VGCC.

Perivascular nerve activity has been linked to local endothelial cell (EC) Ca2+ events through α1‐adrenoreceptor (AR) activation, but the effects of aging on perivascular nerves and EC Ca2+ are unclear. We tested the hypothesis that aging alters local EC Ca2+ signals in vivo. Superfused (pH, 7.4; 36°C) mesenteric arteries (MAs) of anesthetized Young (3–6 month; n=9) and Old (24–26 month; n=5) Cx40BAC‐GCaMP2 mice were imaged (30 frames/s) using spinning disk confocal intravital microscopy. In Young, ECs exhibited spontaneous Ca2+ events [0.31 ± 0.03 Hz; amplitude (F/Fo): 1.31 ± 0.012; duration: 0.11 ± 0.004 s]. In Old, Ca2+ events were nearly absent. Electrical field stimulation (EFS) produced frequency‐dependent MA constriction with Old >; Young by ~20%. Blocking sensory nerves (capsaicin, 10 μM) enhanced MA constriction during EFS by ~20% in Young (P < 0.05) but not Old indicating loss of sensory inhibition of sympathetic vasoconstriction in Old. Cumulative activation (1 nM to 10 μM) of α1ARs (phenylephrine) but not α2ARs (UK14304) exhibited reduced sensitivity (P<0.05) in Old vs. Young. Immunolabeling confirmed sympathetic and sensory nerves were maintained in Old thus functional changes were not due to loss of perivascular innervation. The loss of local EC Ca2+ signals in Old may reflect α1AR desensitization and thereby reduce EC feedback in regulating smooth muscle tone. (NIH R01HL086483, R37HL041026).