Sympathetic and Sensory-Motor Nerves in Peripheral Small Arteries.

Small arteries, which play important roles in controlling blood flow, blood pressure, and capillary pressure, are under nervous influence. Their innervation is predominantly sympathetic and sensory motor in nature, and while some arteries are densely innervated, others are only sparsely so. Innervation of small arteries is a key mechanism in regulating vascular resistance. In the second half of the previous century, the physiology and pharmacology of this innervation were very actively investigated. In the past 10-20 yr, the activity in this field was more limited. With this review we highlight what has been learned during recent years with respect to development of small arteries and their innervation, some aspects of excitation-release coupling, interaction between sympathetic and sensory-motor nerves, cross talk between endothelium and vascular nerves, and some aspects of their role in vascular inflammation and hypertension. We also highlight what remains to be investigated to further increase our understanding of this fundamental aspect of vascular physiology.

Potassium-Channel-Independent Relaxing Influence of Adipose Tissue on Mouse Carotid Artery.

Since the cardiovascular consequences of obesity reportedly vary in different types of obesity, we investigated the influence of adipose tissue from different locales on the phenylephrine-induced tone of the mouse carotid artery. Vessels were mounted in a Mulvany-Halpern-type wire myograph, and adipose tissue, from the back (brown) or mesenteric or inguinal subcutaneous (white), was placed around the artery. Contractile responses to phenylephrine were not affected by brown adipose tissue but were reduced (p < 0.001) by either type of white adipose tissue, with no difference between the 2 locales. The relaxing effect persisted in the presence of the Kv7 channel inhibitor XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone), the KATP channel inhibitor glibenclamide (1 µM), or the KV channel inhibitor 4-amino pyridine (1 mM), as well as after elevation of the extracellular potassium concentration to 30 mM. Contractions of rat carotid artery were equally reduced by mouse and rat subcutaneous adipose tissue. Thus, white, but not brown, adipose tissue reduces the adrenergic contractions of the carotid artery with no differences between the locales of origin, and the effect appears largely independent of potassium channels.

Rats with adenine-induced chronic renal failure develop low-renin, salt-sensitive hypertension and increased aortic stiffness.

Rats with adenine-induced chronic renal failure (A-CRF) develop metabolic and cardiovascular abnormalities resembling those in patients with chronic kidney disease. The aim of this study was to investigate the mechanisms of hypertension in this model and to assess aortic stiffness in vivo. Male Sprague-Dawley rats were equipped with radiotelemetry probes for arterial pressure recordings and received either chow containing adenine or normal control diet. At 7 to 11 wk after study start, blood pressure responses to high NaCl (4%) diet and different pharmacological interventions were analyzed. Aortic pulse wave velocity was measured under isoflurane anesthesia. Baseline 24-h mean arterial pressure (MAP) was 101 ± 10 and 119 ± 9 mmHg in controls and A-CRF animals, respectively (P < 0.01). After 5 days of a high-NaCl diet, MAP had increased by 24 ± 6 mmHg in A-CRF animals vs. 2 ± 1 mmHg in controls (P < 0.001). Candesartan (10 mg/kg by gavage) produced a more pronounced reduction of MAP in controls vs. A-CRF animals (-12 ± 3 vs. -5 ± 5 mmHg, P < 0.05). Aortic pulse wave velocity was elevated in A-CRF rats (5.10 ± 0.51 vs. 4.58 ± 0.17 m/s, P < 0.05). Plasma levels of creatinine were markedly elevated in A-CRF animals (259 ± 46 vs. 31 ± 2 µM, P < 0.001), whereas plasma renin activity was suppressed (0.6 ± 0.5 vs. 12.3 ± 7.3 µg·l(-1)·h(-1), P < 0.001). In conclusion, hypertension in A-CRF animals is characterized by low plasma renin activity and is aggravated by high-NaCl diet, suggesting a pathogenic role for sodium retention and hypervolemia probably secondary to renal insufficiency. Additionally, aortic stiffness was elevated in A-CRF animals as indicated by increased aortic pulse wave velocity.

Vascular function in rats with adenine-induced chronic renal failure.

The aim of the present study was to characterize the function of resistance arteries, and the aorta, in rats with adenine-induced chronic renal failure (A-CRF). Sprague-Dawley rats were randomized to chow with or without adenine supplementation. After 6-10 wk, mesenteric arteries and thoracic aortas were analyzed ex vivo by wire myography. Plasma creatinine concentrations were elevated twofold at 2 wk, and eight-fold at the time of death in A-CRF animals. Ambulatory systolic and diastolic blood pressures measured by radiotelemetry were significantly elevated in A-CRF animals from week 3 and onward. At death, A-CRF animals had anemia, hyperphosphatemia, hyperparathyroidism, and elevated plasma levels of asymmetric dimethylarginine and oxidative stress markers. There were no significant differences between groups in the sensitivity, or maximal response, to ACh, sodium nitroprusside (SNP), norepinephrine, or phenylephrine in either mesenteric arteries or aortas. However, in A-CRF animals, the rate of aortic relaxation was significantly reduced following washout of KCl (both in intact and endothelium-denuded aorta) and in response to ACh and SNP. Also the rate of contraction in response to KCl was significantly reduced in A-CRF animals both in mesenteric arteries and aortas. The media of A-CRF aortas was thickened and showed focal areas of fragmented elastic lamellae and disorganized smooth muscle cells. No vascular calcifications could be detected. These results indicate that severe renal failure for a duration of less than 10 wk in this model primarily affects the aorta and mainly slows the rate of relaxation.

Bestrophin-3 (vitelliform macular dystrophy 2-like 3 protein) is essential for the cGMP-dependent calcium-activated chloride conductance in vascular smooth muscle cells.

Although the biophysical fingerprints (ion selectivity, voltage-dependence, kinetics, etc) of Ca(2+)-activated Cl(-) currents are well established, their molecular identity is still controversial. Several molecular candidates have been suggested; however, none of them has been fully accepted. We have recently characterized a cGMP-dependent Ca(2+)-activated Cl(-) current with unique characteristics in smooth muscle cells. This novel current has been shown to coexist with a "classic" (cGMP-independent) Ca(2+)-activated Cl(-) current and to have characteristics distinct from those previously known for Ca(2+)-activated Cl(-) currents. Here, we suggest that a bestrophin, a product of the Best gene family, is responsible for the cGMP-dependent Ca(2+)-activated Cl(-) current based on similarities between the membrane currents produced by heterologous expressions of bestrophins and the cGMP-dependent Ca(2+)-activated Cl(-) current. This is supported by similarities in the distribution pattern of the cGMP-dependent Ca(2+)-activated Cl(-) current and bestrophin-3 (the product of Best-3 gene) expression in different smooth muscle. Furthermore, downregulation of Best-3 gene expression with small interfering RNA both in cultured cells and in vascular smooth muscle cells in vivo was associated with a significant reduction of the cGMP-dependent Ca(2+)-activated Cl(-) current, whereas the magnitude of the classic Ca(2+)-activated Cl(-) current was not affected. The majority of previous suggestions that bestrophins are a new Cl(-) channel family were based on heterologous expression in cell culture studies. Our present results demonstrate that at least 1 family member, bestrophin-3, is essential for a well-defined endogenous Ca(2+)-activated Cl(-) current in smooth muscles in the intact vascular wall.

Expression of S100A Alarmins in Cord Blood Monocytes Is Highly Associated With Chorioamnionitis and Fetal Inflammation in Preterm Infants.

Background: Preterm infants exposed to chorioamnionitis and with a fetal inflammatory response are at risk for neonatal morbidity and adverse outcome. Alarmins S100A8, S100A9, and S100A12 are expressed by myeloid cells and have been associated with inflammatory activation and monocyte modulation. Aim: To study S100A alarmin expression in cord blood monocytes from term healthy and preterm infants and relate results to clinical findings, inflammatory biomarkers and alarmin protein levels, as well as pathways identified by differentially regulated monocyte genes. Methods: Cord blood CD14+ monocytes were isolated from healthy term (n = 10) and preterm infants (<30 weeks gestational age, n = 33) by MACS technology. Monocyte RNA was sequenced and gene expression was analyzed by Principal Component Analysis and hierarchical clustering. Pathways were identified by Ingenuity Pathway Analysis. Inflammatory proteins were measured by Multiplex ELISA, and plasma S100A proteins by mass spectrometry. Histological chorioamnionitis (HCA) and fetal inflammatory response syndrome (FIRS) were diagnosed by placenta histological examination. Results: S100A8, S100A9, and S100A12 gene expression was significantly increased and with a wider range in preterm vs. term infants. High S100A8 and S100A9 gene expression (n = 17) within the preterm group was strongly associated with spontaneous onset of delivery, HCA, FIRS and elevated inflammatory proteins in cord blood, while low expression (n = 16) was associated with impaired fetal growth and physician-initiated delivery. S100A8 and S100A9 protein levels were significantly lower in preterm vs. term infants, but within the preterm group high S100A gene expression, spontaneous onset of labor, HCA and FIRS were associated with elevated protein levels. One thousand nine hundred genes were differentially expressed in preterm infants with high vs. low S100A alarmin expression. Analysis of 124 genes differentially expressed in S100A high as well as FIRS and HCA groups identified 18 common pathways and S100A alarmins represented major hubs in network analyses. Conclusion: High expression of S100A alarmins in cord blood monocytes identifies a distinct clinical risk group of preterm infants exposed to chorioamnionitis and with a fetal inflammatory response. Gene and pathway analyses suggest that high S100A alarmin expression also affects monocyte function. The connection with monocyte phenotype and inflammation-stimulated S100A expression in other cell types (e.g., neutrophils) warrants further investigation.

RNA sequencing data describing transcriptional changes in aorta of ApoE-/- mice after alpha 7 nicotinic acetylcholine receptor (α7nAChR) stimulation.

This manuscript is a companion paper to Ulleryd M.U. et al., "Stimulation of alpha 7 nicotinic acetylcholine receptor (α7nAChR) inhibits atherosclerosis via immunomodulatory effects on myeloid cells" Atherosclerosis, 2019 [1]. Data shown here include RNA sequencing data from whole aorta of ApoE-/- mice fed high fat diet and treated with the alpha 7 nicotinic acetylcholine receptor (α7nAChR) agonist AZ6983 for 8 weeks using subcutaneously implanted osmotic minipumps. Here we present the top gene networks affected by treatment with AZ6983, as well as the up- and down-regulated genes in aorta after treatment. Further, a URL link to the RNA sequencing datasets submitted to GEO is included.

Interaction between Na+/K+-pump and Na+/Ca2+-exchanger modulates intercellular communication.

Ouabain, a specific inhibitor of the Na(+)/K(+)-pump, has previously been shown to interfere with intercellular communication. Here we test the hypothesis that the communication between vascular smooth muscle cells is regulated through an interaction between the Na(+)/K(+)-pump and the Na(+)/Ca(2+)-exchanger leading to an increase in the intracellular calcium concentration ([Ca(2+)](i)) in discrete areas near the plasma membrane. [Ca(2+)](i) in smooth muscle cells was imaged in cultured rat aortic smooth muscle cell pairs (A7r5) and in rat mesenteric small artery segments simultaneously with force. In A7r5 coupling between cells was estimated by measuring membrane capacitance. Smooth muscle cells were uncoupled when the Na(+)/K(+)-pump was inhibited either by a low concentration of ouabain, which also caused a localized increase of [Ca(2+)](i) near the membrane, or by ATP depletion. Reduction of Na(+)/K(+)-pump activity by removal of extracellular potassium ([K(+)](o)) also uncoupled cells, but only after inhibition of K(ATP) channels. Inhibition of the Na(+)/Ca(2+)-exchange activity by SEA0400 or by a reduction of the equilibrium potential (making it more negative) also uncoupled the cells. Depletion of intracellular Na(+) and clamping of [Ca(2+)](i) at low concentrations prevented the uncoupling. The experiments suggest that the Na(+)/K(+)-pump may affect gap junction conductivity via localized changes in [Ca(2+)](i) through modulation of Na(+)/Ca(2+)-exchanger activity.

Bestrophin-3 Expression in a Subpopulation of Astrocytes in the Neonatal Brain After Hypoxic-Ischemic Injury.

Bestrophin-3, a potential candidate for a calcium-activated chloride channel, recently was suggested to have cell-protective functions. We studied the expression and alternative splicing of bestrophin-3 in neonatal mouse brain and after hypoxic-ischemic (HI) injury and in human neonatal brain samples. HI brain injury was induced in 9-day old mice by unilateral permanent common carotid artery occlusion in combination with exposure to 10% oxygen for 50 min. Endoplasmic reticulum stress was induced by thapsigargin treatment in primary culture of mouse brain astrocytes. We also investigated expression of bestrophin-3 protein in a sample of human neonatal brain tissue. Bestrophin-3 protein expression was detected with immunohistochemical methods and western blot; mRNA expression and splicing were analyzed by RT-PCR. HI induced a brain tissue infarct and a pronounced increase in the endoplasmic reticulum-associated marker CHOP. Three days after HI a population of astrocytes co-expressed bestrophin-3 and nestin in a penumbra-like area of the injured hemisphere. However, total levels of Bestrophin-3 protein in mouse cortex were reduced after injury. Mouse astrocytes in primary culture also expressed bestrophin-3 protein, the amount of which was reduced by endoplasmic reticulum stress. Bestrophin-3 protein was detected in astrocytes in the hippocampal region of the human neonatal brain which had patchy white matter gliosis and neuronal loss in the Sommer's sector of the Ammon's horn (CA1). Analysis of bestrophin-3 mRNA in mouse brain with and without injury showed the presence of two truncated spliced variants, but no full-length mRNA. Total amount of bestrophin-3 mRNA increased after HI, but showed only minor injury-related change. However, the splice variants of bestrophin-3 mRNA were differentially regulated after HI depending on the presence of tissue injury. Our results show that bestrophin-3 is expressed in neonatal mouse brain after injury and in the human neonatal brain with pathology. In mouse brain bestrophin-3 protein is upregulated in a specific astrocyte population after injury and is co-expressed with nestin. Splice variants of bestrophin-3 mRNA respond differently to HI, which might indicate their different roles in tissue injury.

Stimulation of alpha 7 nicotinic acetylcholine receptor (α7nAChR) inhibits atherosclerosis via immunomodulatory effects on myeloid cells.

BACKGROUND AND AIMS: Alpha 7 nicotinic acetylcholine receptor (α7nAChR) stimulation can regulate acute inflammation, and lack of α7nAChR accelerates atherosclerosis in mice. In this study, we aimed to investigate the effects of the novel α7nAChR agonist, AZ6983, on atherosclerosis and assess its possible immunomodulating effects. METHODS: AZ6983 was tested in vitro in LPS-challenged mouse and human blood and in vivo using the acute inflammatory air pouch model. Thereafter, long-term effects of AZ6983 treatment on atherosclerosis and immune responses were assessed in apoE-/- mice after 8 and 12 weeks. Atherosclerosis was investigated in the aortic root and thoracic aorta, serum levels of cytokines were analysed and RNAseq was used to study aortic gene expression. Further, bone-marrow-derived macrophages were used to assess phagocytosis in vitro. RESULTS: α7nAChR activation by AZ6983 decreased pro-inflammatory cytokines in acute stimulations of human and mouse blood in vitro, as well as in vivo using the air pouch model. Treating apoE-/- mice with AZ6983 decreased atherosclerosis by 37-49% and decreased serum cytokine levels. RNAseq analysis of aortae suggested the involvement of several specific myeloid cell functions, including phagocytosis. In line with this, AZ6983 significantly increased phagocytosis in bone marrow-derived macrophages. CONCLUSIONS: This study demonstrates that activation of α7nAChR with AZ6983 inhibits atherosclerosis in apoE-/-mice and that immunomodulating effects on myeloid cells, such as enhanced phagocytosis and suppression of inflammatory cytokines, could be part of the athero-protective mechanisms. The observed anti-inflammatory effect in human blood supports the idea that AZ6983 may decrease disease also in humans.

[Ca2+ ] changes in sympathetic varicosities and Schwann cells in rat mesenteric arteries-Relation to noradrenaline release and contraction.

AIM: This study aimed to assess intracellular Ca2+ dynamics in nerve cells and Schwann cells in isolated rat resistance arteries and determine how these dynamics modify noradrenaline release from the nerves and consequent force development. METHODS: Ca2+ in nerves was assessed with confocal imaging, noradrenaline release with amperometry and artery tone with wire myography. Ca2+ in axons was assessed after loading with Oregon Green 488 BAPTA-1 dextran. In other experiments, arteries were incubated with Calcium Green-1-AM which loads both axons and Schwann cells. RESULTS: Schwann cells but not axons responded with a Ca2+ increase to ATP. Electrical field stimulation of nerves caused a frequency-dependent increase in varicose [Ca2+ ] ([Ca2+ ]v ). ω-conotoxin-GVIA (100 nmol/L) reduced the [Ca2+ ]v transient to 2 and 16 Hz by 60% and 27%, respectively; in contrast ω-conotoxin GVIA inhibited more than 80% of the noradrenaline release and force development at 2 and 16 Hz. The KV channel blocker, 4-aminopyridine (10 µmol/L), increased [Ca2+ ]v , noradrenaline release and force development both in the absence and presence of ω-conotoxin-GVIA. Yohimbine (1 µmol/L) increased both [Ca2+ ]v and noradrenaline release but reduced force development. Acetylcholine (10 µmol/L) caused atropine-sensitive inhibition of [Ca2+ ]v , noradrenaline release and force. In the presence of ω-conotoxin-GVIA, acetylcholine caused a further inhibition of all parameters. CONCLUSION: Modification of [Ca2+ ] in arterial sympathetic axons and Schwann cells was assessed separately. KV 3.1 channels may be important regulators of [Ca2+ ]v , noradrenaline release and force development. Presynaptic adrenoceptor and muscarinic receptor activation modify transmitter release through modification of [Ca2+ ]v .

Antiphase oscillations of endothelium and smooth muscle [Ca2+]i in vasomotion of rat mesenteric small arteries.

The mechanisms leading to vasomotion in the presence of noradrenaline and inhibitors of the sarcoplasmic/endoplasmic reticulum calcium ATPase were investigated in isolated rat mesenteric small arteries. Isobaric diameter and isometric force were measured together with membrane potential in endothelial cells and smooth muscle cells (SMC). Calcium in the endothelial cells and SMC was imaged with confocal microscopy. In the presence of noradrenaline and cyclopiazonic acid, ryanodine-insensitive oscillations in tone were produced. The frequency was about 1 min(-1) and amplitude about 70% of the maximal tone. The amplitude was reduced by indomethacin and increased with L-NAME. Vasomotion was inhibited by nifedipine and by 40 mM potassium. The frequency was increased and amplitude decreased by removal of the endothelium and by application of charybdotoxin and apamin. The vasomotion was associated with in-phase oscillations of membrane potential in endothelial cells and SMC and oscillations of [Ca2+]i that were in near anti-phase. We suggest a working model for the generation of oscillation based on a membrane oscillator where ion channels in both endothelial cells and SMC interact via a current running between the two cell types through myoendothelial gap junctions, which sets up a near anti-phase oscillation of [Ca2+]i in the two cell types.

Investigation of vascular responses in endothelial nitric oxide synthase/cyclooxygenase-1 double-knockout mice: key role for endothelium-derived hyperpolarizing factor in the regulation of blood pressure in vivo.

BACKGROUND: Endothelium-dependent dilatation is mediated by 3 principal vasodilators: nitric oxide (NO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF). To determine the relative contribution of these factors in endothelium-dependent relaxation, we have generated mice in which the enzymes required for endothelial NO and PGI2 production, endothelial NO synthase (eNOS) and cyclooxygenase-1 (COX-1), respectively, have been disrupted (eNOS-/- and COX-1-/- mice). METHODS AND RESULTS: In female mice, the absence of eNOS and COX-1 had no effect on mean arterial blood pressure (BP), whereas BP was significantly elevated in eNOS-/-/COX-1-/- males compared with wild-type controls. Additionally, endothelium-dependent relaxation remained intact in the resistance vessels of female mice and was associated with vascular smooth muscle hyperpolarization; however, these responses were profoundly suppressed in arteries of male eNOS-/-/COX-1-/- animals. Similarly, the endothelium-dependent vasodilator bradykinin produced dose-dependent hypotension in female eNOS-/-/COX-1-/- animals in vivo but had no effect on BP in male mice. CONCLUSIONS: These studies indicate that EDHF is the predominant endothelium-derived relaxing factor in female mice, whereas NO and PGI2 are the predominant mediators in male mice. Moreover, the gender-specific prevalence of EDHF appears to underlie the protection of female eNOS-/-/COX-1-/- mice against hypertension.

A cyclic GMP-dependent calcium-activated chloride current in smooth-muscle cells from rat mesenteric resistance arteries.

We have previously demonstrated the presence of a cyclic GMP (cGMP)-dependent calcium-activated inward current in vascular smooth-muscle cells, and suggested this to be of importance in synchronizing smooth-muscle contraction. Here we demonstrate the characteristics of this current. Using conventional patch-clamp technique, whole-cell currents were evoked in freshly isolated smooth-muscle cells from rat mesenteric resistance arteries by elevation of intracellular calcium with either 10 mM caffeine, 1 microM BAY K8644, 0.4 microM ionomycin, or by high calcium concentration (900 nM) in the pipette solution. The current was found to be a calcium-activated chloride current with an absolute requirement for cyclic GMP (EC50 6.4 microM). The current could be activated by the constitutively active subunit of PKG. Current activation was blocked by the protein kinase G antagonist Rp-8-Br-PET-cGMP or with a peptide inhibitor of PKG, or with the nonhydrolysable ATP analogue AMP-PNP. Under biionic conditions, the anion permeability sequence of the channel was SCN- > Br- > I- > Cl- > acetate > F- >> aspartate, but the conductance sequence was I- > Br- > Cl- > acetate > F- > aspartate = SCN-. The current had no voltage or time dependence. It was inhibited by nickel and zinc ions in the micromolar range, but was unaffected by cobalt and had a low sensitivity to inhibition by the chloride channel blockers niflumic acid, DIDS, and IAA-94. The properties of this current in mesenteric artery smooth-muscle cells differed from those of the calcium-activated chloride current in pulmonary myocytes, which was cGMP-independent, exhibited a high sensitivity to inhibition by niflumic acid, was unaffected by zinc ions, and showed outward current rectification as has previously been reported for this current. Under conditions of high calcium in the patch-pipette solution, a current similar to the latter could be identified also in the mesenteric artery smooth-muscle cells. We conclude that smooth-muscle cells from rat mesenteric resistance arteries have a novel cGMP-dependent calcium-activated chloride current, which is activated by intracellular calcium release and which has characteristics distinct from other calcium-activated chloride currents.

Vasomotion: cellular background for the oscillator and for the synchronization of smooth muscle cells.

1. Vasomotion is the oscillation of vascular tone with frequencies in the range from 1 to 20 min(-1) seen in most vascular beds. The oscillation originates in the vessel wall and is seen both in vivo and in vitro. 2. Recently, our ideas on the cellular mechanisms responsible for vasomotion have improved. Three different types of cellular oscillations have been suggested. One model has suggested that oscillatory release of Ca2+ from intracellular stores is important (the oscillation is based on a cytosolic oscillator). A second proposed mechanism is an oscillation originating in the sarcolemma (a membrane oscillator). A third mechanism is based on an oscillation of glycolysis (metabolic oscillator). For the two latter mechanisms, only limited experimental evidence is available. 3. To understand vasomotion, it is important to understand how the cells synchronize. For the cytosolic oscillators synchronization may occur via activation of Ca2+-sensitive ion channels by oscillatory Ca2+ release. The ensuing membrane potential oscillation feeds back on the intracellular Ca2+ stores and causes synchronization of the Ca2+ release. While membrane oscillators in adjacent smooth muscle cells could be synchronized through the same mechanism that sets up the oscillation in the individual cells, a mechanism to synchronize the metabolic-based oscillators has not been suggested. 4. The interpretation of the experimental observations is supported by theoretical modelling of smooth muscle cells behaviour, and the new insight into the mechanisms of vasomotion has the potential to provide tools to investigate the physiological role of vasomotion.

NO contributes to EDHF-like responses in rat small arteries: a role for NO stores.

OBJECTIVES: Responses to EDHF are usually characterised in the presence of nitric oxide synthase (NOS) and cyclooxygenase (COX) inhibitors. The contribution of NO to endothelium-dependent relaxation in the presence of NOS inhibitors was assessed using NO scavengers with the objective of testing (i) whether any residual NO produces endothelium-dependent relaxation in a manner similar to EDHF and (ii) to identify the source of the residual NO. METHODS: Small rat hepatic and mesenteric arteries were mounted in a tension myograph for either isometric or membrane potential measurements. RESULTS: Relaxation to ACh was unaffected by pre-treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 300 microM), and indomethacin (Indo, 5 microM) in the absence or presence of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 microM), nitro-L-arginine (300 microM) or L-nitro-mono-methyl-arginine (L-NMMA, 300 microM). Addition of OxyHb (20 microM) or carboxy-PTIO (300 microM) produced a significant suppression of ACh-induced relaxations ( approximately 40%). In L-NAME+Indo treated arteries ACh-induced hyperpolarisation (delta16.3+/-2.1 mV, n=8) was significantly suppressed with the addition of OxyHb (Delta10.2+/-1.6 mV, n=12). ACh-induced relaxation, in the presence of L-NAME+Indo+OxyHb, was abolished by raised extracellular K(+), or the combination of charybdotoxin (CTX, 100 nM)+apamin (100 nM). In contrast whilst L-NAME+indo+barium+ouabain suppressed ACh-induced relaxation, the presence of OxyHb had no additional effect. Ultraviolet light induced a relaxation in arteries treated with L-NMMA+Indo (37.0+/-5.2%, n=9) which was sensitive to OxyHb (15.2+/-10.9%, n=4), and barium+ouabain (6.39+/-2.7%, n=4), but not CTX+apamin (37.8+/-2.4%, n=4). CONCLUSIONS: These findings suggest that NO contributes significantly to the "EDHF-like" response seen in rat small arteries and that the source of this NO may be preformed vascular stores.

Brain barrier properties and cerebral blood flow in neonatal mice exposed to cerebral hypoxia-ischemia.

Insults to the developing brain often result in irreparable damage resulting in long-term deficits in motor and cognitive functions. The only treatment today for hypoxic-ischemic encephalopathy (HIE) in newborns is hypothermia, which has limited clinical benefit. We have studied changes to the blood-brain barriers (BBB) as well as regional cerebral blood flow (rCBF) in a neonatal model of HIE to further understand the underlying pathologic mechanisms. Nine-day old mice pups, brain roughly equivalent to the near-term human fetus, were subjected to hypoxia-ischemia. Hypoxia-ischemia increased BBB permeability to small and large molecules within hours after the insult, which normalized in the following days. The opening of the BBB was associated with changes to BBB protein expression whereas gene transcript levels were increased showing direct molecular damage to the BBB but also suggesting compensatory mechanisms. Brain pathology was closely related to reductions in rCBF during the hypoxia as well as the areas with compromised BBB showing that these are intimately linked. The transient opening of the BBB after the insult is likely to contribute to the pathology but at the same time provides an opportunity for therapeutics to better reach the infarcted areas in the brain.

Junctional and nonjunctional effects of heptanol and glycyrrhetinic acid derivates in rat mesenteric small arteries.

1 Heptanol, 18alpha-glycyrrhetinic acid (18alphaGA) and 18beta-glycyrrhetinic acid (18betaGA) are known blockers of gap junctions, and are often used in vascular studies. However, actions unrelated to gap junction block have been repeatedly suggested in the literature for these compounds. We report here the findings from a comprehensive study of these compounds in the arterial wall. 2 Rat isolated mesenteric small arteries were studied with respect to isometric tension (myography), [Ca2+]i (Ca(2+)-sensitive dyes), membrane potential and--as a measure of intercellular coupling--input resistance (sharp intracellular glass electrodes). Also, membrane currents (patch-clamp) were measured in isolated smooth muscle cells (SMCs). Confocal imaging was used for visualisation of [Ca2+]i events in single SMCs in the arterial wall. 3 Heptanol (150 microm) activated potassium currents, hyperpolarised the membrane, inhibited the Ca2+ current, and reduced [Ca2+]i and tension, but had little effect on input resistance. Only at concentrations above 200 microm did heptanol elevate input resistance, desynchronise SMCs and abolish vasomotion. 4 18betaGA (30 microm) not only increased input resistance and desynchronised SMCs but also had nonjunctional effects on membrane currents. 18alphaGA (100 microm) had no significant effects on tension, [Ca2+]i, total membrane current and synchronisation in vascular smooth muscle. 5 We conclude that in mesenteric small arteries, heptanol and 18betaGA have important nonjunctional effects at concentrations where they have little or no effect on intercellular communication. Thus, the effects of heptanol and 18betaGA on vascular function cannot be interpreted as being caused only by effects on gap junctions. 18alphaGA apparently does not block communication between SMCs in these arteries, although an effect on myoendothelial gap junctions cannot be excluded.

KATP-channel-induced vasodilation is modulated by the Na,K-pump activity in rabbit coronary small arteries.

The purpose of the study was to evaluate the importance of the Na,K-pump in relaxations induced by K(ATP)-channel openers in rabbit coronary small arteries. Arterial segments were mounted in myographs for recording of isometric tension. Whole-cell patch clamp was used to assess K(ATP)-channel currents in isolated smooth muscle cells from the arteries. In arteries preconstricted with the thromboxane A(2) analogue U46619 pinacidil and cromakalim induced concentration-dependent relaxations. In arteries preconstricted with potassium (124 mM) only high concentrations of pinacidil had a small relaxant effect. In arteries preconstricted with U46619 pinacidil-induced relaxations were unaffected by pretreatment with N(omega)-nitro-L-arginine (L-NNA) and only slightly reduced after mechanical removal of the endothelium. Pinacidil induced relaxations were not significantly affected by 1 microM glibenclamide. However, the relaxations were partly inhibited in potassium-free media and by 1 microM ouabain. In contrast, the concentration-dependent relaxation to cromakalim was partly blocked by 1 microM glibenclamide and partly by 1 microM ouabain and when both drugs were present the inhibition increased. Ouabain (1 microM) and glibenclamide (1 microM) each partly inhibited an ATP-sensitive current induced by pinacidil and cromakalim. In the presence of both inhibitors a greater inhibition was seen. When the solution in the patch pipette was sodium-free the current was reduced and ouabain had no effect. The study suggests that the relaxation to cromakalim and most likely pinacidil is mediated through opening of K(ATP) channels. Inhibition of the Na,K-pump, however, may change the local environment for the K(ATP) channels (i.e. increases the ATP/ADPratio and/or decreases the transmembrane potassium gradient), which partly prevents the activation of the K(ATP)-channel current.

Endothelial function in a mouse model of myeloperoxidase deficiency.

Myeloperoxidase (MPO) activity is suggested to reduce the function of vascular nitric oxide, thereby contributing to endothelial dysfunction, although data in rodents are inconclusive. We examined vascular contractile and relaxant responses in MPO-deficient (MPO(-/-)) and wild-type mice to investigate the role for myeloperoxidase in the development of endothelial dysfunction. Carotid and saphenous arteries were taken from 8-month-old mice and studied in a myograph. Responses of carotid arteries to phenylephrine, high potassium, or acetylcholine (Ach) were statistically not different from controls. Treatment with lipopolysaccharide (LPS; to enhance endothelial dysfunction) reduced responses to Ach in MPO(-/-) but did not affect responses in wild-type. In response to high concentrations of Ach, carotid arteries responded with transient contractions, which were not different between the groups and not affected by LPS treatment. Saphenous arteries from MPO(-/-) had smaller normalized diameters and developed less contractile force. Vessels from MPO(-/-) were less sensitive to Ach than controls. These data suggest that mature MPO-deficient mice do not show enhanced endothelial function compared to wild-type mice, even when provoked with LPS treatment. The EDHF response appears to be reduced in MPO deficiency.