Rebecca E. Haddock

Rhythmicity in arterial smooth muscle

Many arteries and arterioles exhibit rhythmical contractions which are synchronous over considerable distances. This vasomotion is likely to assist in tissue perfusion especially during periods of altered metabolism or perfusion pressure. While the mechanism underlying vascular rhythmicity has been investigated for many years, it has only been recently, with the advent of imaging techniques for visualizing intracellular calcium release, that significant advances have been made. These methods, when combined with mechanical and electrophysiological recordings, have demonstrated that the rhythm depends critically on calcium released from intracellular stores within the smooth muscle cells and on cell coupling via gap junctions to synchronize oscillations in calcium release amongst adjacent cells. While these factors are common to all vessels studied to date, the contribution of voltage‐dependent channels and the endothelium varies amongst different vessels. The basic mechanism for rhythmical activity in arteries thus differs from its counterpart in non‐vascular smooth muscle, where specific networks of pacemaker cells generate electrical potentials which drive activity within the otherwise quiescent muscle cells.

WHAT'S WHERE AND WHY AT A VASCULAR MYOENDOTHELIAL MICRODOMAIN SIGNALLING COMPLEX

1 Modulation of vascular cell calcium is critical for the control of vascular tone, blood flow and pressure. 2 Specialized microdomain signalling sites associated with calcium modulation are present in vascular smooth muscle cells, where spatially localized channels and calcium store receptors interact functionally. Anatomical studies suggest that such sites are also present in endothelial cells. 3 The characteristics of these sites near heterocellular myoendothelial gap junctions (MEGJs) are described, focusing on rat mesenteric artery. The MEGJs enable current and small molecule transfer to coordinate arterial function and are thus critical for endothelium‐derived hyperpolarization, regulation of smooth muscle cell diameter in response to contractile stimuli and vasomotor conduction over distance. 4 Although MEGJs occur on endothelial cell projections within internal elastic lamina (IEL) holes, not all IEL holes have MEGJ‐related projections (approximately 0–50% of such holes have MEGJ‐related projections, with variations occurring within and between vessels, species, strains and disease). 5 In rat mesenteric, saphenous and caudal cerebellar artery and hamster cheek pouch arteriole, but not rat middle cerebral artery or cremaster arteriole, intermediate conductance calcium‐activated potassium channels (IKCa) localize to endothelial cell projections. 6 Rat mesenteric artery MEGJ connexins and IKCa are in close spatial association with endothelial cell inositol 1,4,5‐trisphosphate receptors and endoplasmic reticulum. 7 Data suggest a relationship between spatially associated endothelial cell ion channels and calcium stores in modulation of calcium release and action. Differences in spatial relationships between ion channels and calcium stores in different vessels reflect heterogeneity in vasomotor function, representing a selective target for the control of endothelial and vascular function.

Differential activation of ion channels by inositol 1,4,5-trisphosphate (IP3)- and ryanodine-sensitive calcium stores in rat basilar artery vasomotion.

Spontaneous, rhythmical contractions, or vasomotion, can be recorded from cerebral vessels under both normal physiological and pathophysiological conditions. Using electrophysiology to study changes in membrane potential, the ratiometric calcium indicator Fura‐2 AM to study changes in [Ca2+]i in both the arterial wall and in individual smooth muscle cells (SMCs), and video microscopy to study changes in vessel diameter, we have investigated the cellular mechanisms underlying vasomotion in the juvenile rat basilar artery. During vasomotion, rhythmical oscillations in both membrane potential and [Ca2+]i were found to precede rhythmical contractions. Nifedipine depolarized SMCs and abolished rhythmical contractions and depolarizations. [Ca2+]i oscillations in the arterial wall became reduced and irregular, while [Ca2+]i oscillations in adjacent SMCs were no longer synchronized. BAPTA‐AM, thapsigargin and U73122 hyperpolarized SMCs, relaxed the vessel, decreased basal calcium levels and abolished vasomotion. Chloride substitution abolished rhythmical activity, depolarized SMCs, increased basal calcium levels and constricted the vessel, while niflumic acid and DIDS abolished vasomotion. Ryanodine, charybdotoxin and TRAM‐34, but not iberiotoxin, 4‐aminopyridine or apamin, each depolarized SMCs and increased the frequency of rhythmical depolarizations and [Ca2+]i oscillations. We conclude that vasomotion in the basilar artery depends on the release of intracellular calcium from IP3 (inositol 1,4,5,‐trisphosphate)‐sensitive stores which activates calcium‐dependent chloride channels to depolarize SMCs. Depolarization in turn activates voltage‐dependent calcium channels, synchronizing contractions of adjacent cells through influx of extracellular calcium. Subsequent calcium‐induced calcium release from ryanodine‐sensitive stores activates an intermediate conductance potassium channel, hyperpolarizing the SMCs and providing a negative feedback pathway for regeneration of the contractile cycle.

Heterogeneity in function of small artery smooth muscle BKCa: involvement of the β1‐subunit

Arteriolar myogenic vasoconstriction occurs when increased stretch or membrane tension leads to smooth muscle cell depolarization and opening of voltage‐gated Ca2+ channels. To prevent positive feedback and excessive pressure‐induced vasoconstriction, studies in cerebral artery smooth muscle have suggested that activation of large conductance, Ca2+‐activated K+ channels (BKCa) provides an opposing hyperpolarizing influence reducing Ca2+ channel activity. We have hypothesized that this mechanism may not equally apply to all vascular beds. To establish the existence of such heterogeneity in vascular reactivity, studies were performed on rat vascular smooth muscle (VSM) cells from cremaster muscle arterioles and cerebral arteries. Whole cell K+ currents were determined at pipette [Ca2+] of 100 nm or 5 μm in the presence and absence of the BKCa inhibitor, iberiotoxin (IBTX; 0.1 μm). Similar outward current densities were observed for the two cell preparations at the lower pipette Ca2+ levels. At 5 μm Ca2+, cremaster VSM showed a significantly (P < 0.05) lower current density compared to cerebral VSM (34.5 ± 1.9 vs 45.5 ± 1.7 pA pF−1 at +70 mV). Studies with IBTX suggested that the differences in K+ conductance at 5 μm intracellular [Ca2+] were largely due to activity of BKCa. 17β‐Oestradiol (1 μm), reported to potentiate BKCa current via the channels β‐subunit, caused a greater effect on whole cell K+ currents in cerebral vessel smooth muscle cells (SMCs) compared to those of cremaster muscle. In contrast, the α‐subunit‐selective BKCa opener, NS‐1619 (20 μm), exerted a similar effect in both preparations. Spontaneously transient outward currents (STOCs) were more apparent (frequency and amplitude) and occurred at more negative membrane potentials in cerebral compared to cremaster SMCs. Also consistent with decreased STOC activity in cremaster SMCs was an absence of detectable Ca2+ sparks (0 of 76 cells) compared to that in cerebral SMCs (76 of 105 cells). Quantitative PCR showed decreased mRNA expression for the β1 subunit and a decrease in the β 1: α ratio in cremaster arterioles compared to cerebral vessels. Similarly, cremaster arterioles showed a decrease in total BKCa protein and the β 1: α‐subunit ratio. The data support vascular heterogeneity with respect to the activity of BKCa in terms of both β‐subunit regulation and interaction with SR‐mediated Ca2+ signalling.

Obesity Up-Regulates Intermediate Conductance Calcium-Activated Potassium Channels and Myoendothelial Gap Junctions to Maintain Endothelial Vasodilator Function

The mechanisms involved in altered endothelial function in obesity-related cardiovascular disease are poorly understood. This study investigates the effect of chronic obesity on endothelium-dependent vasodilation and the relative contribution of nitric oxide (NO), calcium-activated potassium channels (KCa), and myoendothelial gap junctions (MEGJs) in the rat saphenous artery. Obesity was induced by feeding rats a cafeteria-style diet (∼30 kJ as fat) for 16 to 20 weeks, with this model reflecting human dietary obesity etiology. Age- and sex-matched controls received standard chow (∼12 kJ as fat). Endothelium-dependent vasodilation was characterized in saphenous arteries by using pressure myography with pharmacological intervention, Western blotting, immunohistochemistry, and ultrastructural techniques. In saphenous artery from control, acetylcholine (ACh)-mediated endothelium-dependent vasodilation was blocked by NO synthase and soluble guanylate cyclase inhibition, whereas in obese rats, the ACh response was less sensitive to such inhibition. Conversely, the intermediate conductance KCa (IKCa) blocker 1-[(2-chlorophenyl)diphenyl-methyl]-1H pyrazole attenuates ACh-mediated dilation in obese, but not control, vessels. In a similar manner, putative gap junction block with carbenoxolone increased the pEC50 for ACh in arteries from obese, but not control, rats. IK1 protein and MEGJ expression was up-regulated in the arteries of obese rats, an observation absent in control. Addition of the small conductance KCa blocker apamin had no effect on ACh-mediated dilation in either control or obese rat vessels, consistent with unaltered SK3 expression. Up-regulation of distinct IKCa- and gap junction-mediated pathways at myoendothelial microdomain sites, key mechanisms for endothelial-derived hyperpolarization-type activity, maintains endothelium-dependent vasodilation in diet-induced obese rat saphenous artery. Plasticity of myoendothelial coupling mechanisms represents a significant potential target for therapeutic intervention.

Diet-Induced Obesity Impairs Endothelium-Derived Hyperpolarization via Altered Potassium Channel Signaling Mechanisms

Background The vascular endothelium plays a critical role in the control of blood flow. Altered endothelium-mediated vasodilator and vasoconstrictor mechanisms underlie key aspects of cardiovascular disease, including those in obesity. Whilst the mechanism of nitric oxide (NO)-mediated vasodilation has been extensively studied in obesity, little is known about the impact of obesity on vasodilation to the endothelium-derived hyperpolarization (EDH) mechanism; which predominates in smaller resistance vessels and is characterized in this study. Methodology/Principal Findings Membrane potential, vessel diameter and luminal pressure were recorded in 4th order mesenteric arteries with pressure-induced myogenic tone, in control and diet-induced obese rats. Obesity, reflecting that of human dietary etiology, was induced with a cafeteria-style diet (∼30 kJ, fat) over 16–20 weeks. Age and sexed matched controls received standard chow (∼12 kJ, fat). Channel protein distribution, expression and vessel morphology were determined using immunohistochemistry, Western blotting and ultrastructural techniques. In control and obese rat vessels, acetylcholine-mediated EDH was abolished by small and intermediate conductance calcium-activated potassium channel (SKCa/IKCa) inhibition; with such activity being impaired in obesity. SKCa-IKCa activation with cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) and 1-ethyl-2-benzimidazolinone (1-EBIO), respectively, hyperpolarized and relaxed vessels from control and obese rats. IKCa-mediated EDH contribution was increased in obesity, and associated with altered IKCa distribution and elevated expression. In contrast, the SKCa-dependent-EDH component was reduced in obesity. Inward-rectifying potassium channel (Kir) and Na+/K+-ATPase inhibition by barium/ouabain, respectively, attenuated and abolished EDH in arteries from control and obese rats, respectively; reflecting differential Kir expression and distribution. Although changes in medial properties occurred, obesity had no effect on myoendothelial gap junction density. Conclusion/Significance In obese rats, vasodilation to EDH is impaired due to changes in the underlying potassium channel signaling mechanisms. Whilst myoendothelial gap junction density is unchanged in arteries of obese compared to control, increased IKCa and Na+/K+-ATPase, and decreased Kir underlie changes in the EDH mechanism.

Voltage independence of vasomotion in isolated irideal arterioles of the rat

The cellular mechanisms underlying vasomotion of irideal arterioles from juvenile rats have been studied using electrophysiological methods, ratiometric calcium measurements and video microscopy. Vasomotion was not affected by removal of the endothelium. Spontaneous contractions were preceded by spontaneous depolarizations. Both were abolished by the intracellular calcium chelator, BAPTA AM (20 μm), but not by ryanodine (10 μm), suggesting a dependence on the cyclical release of calcium from intracellular stores, other than those operated by ryanodine receptors. Oscillations were little changed when the membrane potential of short segments of arteriole was either depolarized or hyperpolarized. When the segments were voltage clamped, oscillating inward currents were recorded, indicating that the changes in membrane potential were voltage independent. Vasomotion was preceded by intracellular calcium oscillations and both were abolished by inhibitors of phospholipase C (U73122, 10 μm), phospholipase A2 (AACOCF3, 30 μm) and protein kinase C (chelerythrine chloride, 5 μm, and myristoylated protein kinase C peptide, 10 μm). Inhibition of vasomotion by the dual lipoxygenase and cyclo‐oxygenase inhibitor, NDGA (10 μm), the lipoxygenase inhibitor, ETI (1 μm) but not by the cyclo‐oxygenase inhibitors, aspirin (10 μm) and indomethacin (10 μm), or the cytochrome P450 inhibitor 17‐ODYA (10 μm), suggested an involvement of the lipoxygenase pathway. The observations suggest that vasomotion of iris arterioles is voltage independent and results from the cyclical release of calcium from IP3‐sensitive stores which are activated by cross talk between the phospholipase C and phospholipase A2 pathways in vascular smooth muscle.

394 NGF SYNTHESIS BY INFLAMMATORY CELLS PROMOTES SYMPATHETIC HYPERINNERVATION IN OBESITY

Background and objective: Chronic sympathetic hyperactivity is associated with obesity and with the development of hypertension. Sympathetic overdrive in hypertension occurs as a result of increases in the density of the sympathetic perivascular nerve plexus, consequent to elevated levels of nerve growth factor (NGF). It is unclear whether increased levels of NGF and changes in sympathetic nerve density are associated with hypertension in the obese. Design and Methods: C57BL/6 mice were fed a normal or high fat diet. Mesenteric arteries were isolated and pressurized. Perivascular nerves were activated by electrical field stimulation and vessel diameter examined with videomicroscopy. Inflammatory cell recruitment was determined using light microscopy and tissue sections stained with haemotoxylin and eosin. Immunohistochemistry using antibodies against NGF and synaptic vesicles and confocal microscopy were used to examine arterial NGF expression and perivascular nerve density. Sympathetic adrenergic nerves were identified using the Faglu method. Results: Obesity increased the amplitude vasoconstriction evoked by 1 Hz repetitive nerve stimulation (P < 0.05). Quantitative analysis showed that the density of sympathetic nerves over arteries taken from obese animals was increased (P < 0.05). Obesity had no apparent effect on NGF expression within vascular smooth muscle cells, but was associated with an increased number of inflammatory cells within the adventitia and these cells were shown to express NGF. Conclusions: Nerve-mediated vasoconstriction is augmented in obesity as a result of increased density of the sympathetic perivascular nerve plexus over mesenteric arteries. Changes occur due to increased numbers of inflammatory cells and production of inflammatory cell NGF.