Alison M. Gurney

Kit Positive Cells In The Guinea Pig Bladder

PURPOSEnWe describe the presence of interstitial cells of Cajal (ICC) throughout the wall of the guinea pig bladder.nnnMATERIALS AND METHODSnBladders obtained from male guinea pigs were prepared for immunohistochemical investigations using various primary antibodies, including the specific ICC marker c-kit (Gibco BRL, Grand Island, New York). Enzymatically dispersed cells with a branched morphology were identified as ICC using anti-c-kit. They were loaded with fluo-4acetoxymethyl (Molecular Probes, Eugene, Oregon) and studied using confocal laser scanning microscopy.nnnRESULTSnAnti-c-kit labeling demonstrated that ICC were oriented in parallel with the smooth muscle bundles that run diagonally throughout the bladder. Double labeling with anti-smooth muscle myosin (Sigma Chemical Co., St. Louis, Missouri) revealed that ICC were located on the boundary of smooth muscle bundles. When anti-c-kit was used in combination with the general neuronal antibody protein gene product 9.5 (Ultraclone Ltd., Isle of Wight, United Kingdom) or anti-neuronal nitric oxide synthase, it was noted that there was a close association between nerves and ICC. Enzymatic dissociation of cells from tissue pieces yielded a heterogeneous population of cells containing typical spindle-shaped smooth muscle cells and branched cells resembling ICC from other preparations. The latter could be identified immunohistochemically as ICC using anti-c-kit, whereas the majority of spindle-shaped cells were not Kit positive. Branched cells responded to the application of carbachol by firing Ca2+ waves and they were often spontaneously active.nnnCONCLUSIONSnICC are located on the boundary of smooth muscle bundles in the guinea pig bladder. They fire Ca2+ waves in response to cholinergic stimulation and can be spontaneously active, suggesting that they could act as pacemakers or intermediaries in the transmission of nerve signals to smooth muscle cells.

In situ characterization of CD4 T cell behavior in mucosal and systemic lymphoid tissues during the induction of oral priming and tolerance

The behavior of antigen-specific CD4+ T lymphocytes during initial exposure to antigen probably influences their decision to become primed or tolerized, but this has not been examined directly in vivo. We have therefore tracked such cells in real time, in situ during the induction of oral priming versus oral tolerance. There were marked contrasts with respect to rate and type of movement and clustering between naive T cells and those exposed to antigen in immunogenic or tolerogenic forms. However, the major difference when comparing tolerized and primed T cells was that the latter formed larger and longer-lived clusters within mucosal and peripheral lymph nodes. This is the first comparison of the behavior of antigen-specific CD4+ T cells in situ in mucosal and systemic lymphoid tissues during the induction of priming versus tolerance in a physiologically relevant model in vivo.

Regulation of the resting potential of rabbit pulmonary artery myocytes by a low threshold, O2-sensing potassium current*

The contributions of specific K+ currents to the resting membrane potential of rabbit isolated, pulmonary artery myocytes, and their modulation by hypoxia, were investigated by use of the whole‐cell, patch‐clamp technique. In the presence of 10 μm glibenclamide the resting potential (−50±4 mV, n=18) was unaffected by 10 μm tetraethylammonium ions, 200 nm charybdotoxin, 200 nm iberiotoxin, 100 μm ouabain or 100 μm digitoxin. The negative potential was therefore maintained without ATP‐sensitive (KATP) or large conductance Ca2+‐sensitive (BKCa) K channels, and without the Na+‐K+ATPase. The resting potential, the delayed rectifier current (IK(V)) and the A‐like K+ current (IK(A)) were all reduced in a concentration‐dependent manner by 4‐aminopyridine (4‐AP) and by quinine. 4‐AP was equally potent at reducing the resting potential and IK(V), 10 mm causing depolarization from −44 mV to −22 mV with accompanying inhibition of IK(V) by 56% and IK(A) by 79%. In marked contrast, the effects of quinine on resting potential were poorly correlated with its effects on both IK(A) and IK(V). At 10 mm, quinine reduced IK(V) and IK(A) by 47% and 38%, respectively, with no change in the resting potential. At 100 μm, both currents were almost abolished while the resting potential was reduced <50%. Raising the concentration to 1 mm had little further effect on IK(A) or IK(V), but essentially abolished the resting potential. Reduction of the resting potential by quinine was correlated with inhibition of a voltage‐gated, low threshold, non‐inactivating K+ current, IK(N). Thus, 100 μm quinine reduced both IK(N) and the resting potential by around 50%. The resting membrane potential was the same whether measured after clamping the cell at −80 mV, or immediately after a prolonged period of depolarization at 0 mV, which inactivated IK(A) and IK(V), but not IK(N). When exposed to a hypoxic solution, the O2 tension near the cell fell from 125±6 to 14±2 mmHg (n=20), resulting in a slow depolarization of the myocyte membrane to −35±3 mV (n=16). The depolarization occurred without a change in the amplitude of IK(V) or IK(A), but it was accompanied by 60% inhibition of IK(N) at 0 mV. Our findings suggest that the resting potential of rabbit pulmonary artery myocytes depends on IK(N), and that inhibition of IK(N) may mediate the depolarization induced by hypoxia.

Influence of chronic hypoxia on the contributions of non-inactivating and delayed rectifier K currents to the resting potential and tone of rat pulmonary artery smooth muscle

Exposing rats to chronic hypoxia increased the 4‐aminopyridine (4‐AP) sensitivity of pulmonary arteries. 1 mm 4‐AP caused smooth muscle cell depolarization and contraction in arteries from hypoxic rats, but had little effect in age‐matched controls. Chronic hypoxia downregulated delayed rectifier K+ current (IK(V)), which was nearly 50% blocked by 1 mm 4‐AP, and non‐inactivating K+ current (IK(N)), which was little affected by 1 mm 4‐AP. The results suggest that IK(N) determines resting potential in control rats and that its downregulation following hypoxia leads to depolarization, which activates IK(V) and increases its contribution to resting potential. The hypoxia‐induced increase in 4‐AP sensitivity thus reflects a switch in the major K+ current determining resting potential, from IK(N) to IK(V). This has important implications for the actions and specificity of pulmonary vasodilator drugs.

Reflection/transmission confocal microscopy characterization of single-crystal diamond microlens arrays

Using the method of photoresist reflow and inductively coupled plasma dry etching, we have fabricated microlens arrays in type-IIa natural single-crystal diamond, with diameters down to 10 μm. The surface profile of the microlenses was characterized by atomic force microscopy and was found to match well with a spherical shape, with a surface roughness of better than 1.2 nm. To characterize the optical properties of these diamond microlens arrays, a laser scanning reflection/transmission confocal microscopy technique has been developed. This technique enabled the surface profile of the microlenses to be measured simultaneously with optical parameters including focal length and spot size, opening up an application area for confocal microscopy.

Mechanisms of endothelial dysfunction after ionized radiation: selective impairment of the nitric oxide component of endothelium-dependent vasodilation

Gamma radiation impairs vascular function, leading to the depression of endothelium‐dependent vasodilatation. Loss of the nitric oxide (NO) pathway has been implicated, but little is known about radiation effects on other endothelial mediators. This study investigated the mechanisms of endothelial dysfunction in rabbits subjected to whole‐body irradiation from a cobalt60 source. The endothelium‐dependent relaxation of rabbit aorta evoked by acetylcholine (ACh) or A23187 was impaired in a dose‐dependent manner by irradiation at 2 Gy or above. Inhibition was evident 9 days post‐irradiation and persisted over the 30 day experimental period. Endothelium‐independent responses to glyceryl trinitrate (GTN), sodium nitroprusside (SNP) and 3‐morpholino‐sydnonimine (SIN‐1) were suppressed over a similar dose range at 7–9 days post‐irradiation, but recovered fully by 30 days post‐irradiation. In healthy vessels, ACh‐induced relaxation was inhibited by L‐Nω‐nitroarginine (L‐NA; 3×10−4 M) and charybdotoxin (10−8 M) plus apamin (10−6 M) but resistant to indomethacin, indicating the involvement of NO and endothelium‐derived hyperpolarizing factor (EDHF). Supporting this, ACh caused smooth muscle hyperpolarization that was reduced by L‐NA and charybdotoxin plus apamin. In irradiated vessels, responses to ACh were insensitive to L‐NA but abolished by charybdotoxin plus apamin, indicating selective loss of NO‐mediated relaxation. In animals treated shortly after irradiation with the antioxidant, α‐tocopherol acetate, the NO‐dependent relaxation was restored without effect on the EDHF‐dependent component. The results imply that radiation selectively impairs the NO pathway as a consequence of oxidative stress, while EDHF is able to maintain endothelium‐dependent relaxation at a reduced level.

Potassium Channels Underlying The Resting Potential Of Pulmonary Artery Smooth Muscle Cells

1. The molecular identity of the K channels giving rise to the negative membrane potential of pulmonary artery smooth muscle cells has yet to be determined.

Multiple sites of oxygen sensing and their contributions to hypoxic pulmonary vasoconstriction.

Oxygen sensing by the pulmonary vasculature is important for the regulation of vessel tone and the matching of lung perfusion to ventilation. Airways hypoxia is a major stimulus for vasoconstriction, which diverts blood from hypoxic alveoli to better ventilated areas of the lung. Several hypotheses have emerged to explain how pulmonary arteries sense a decrease in oxygen and mediate hypoxic pulmonary vasoconstriction (HPV). They differ mainly in where they place the main site of HPV: in the endothelial or smooth muscle cells of the artery wall. HPV probably results from synergistic actions on both cell types, but it can proceed in the absence of endothelium, suggesting that the primary oxygen sensor is the smooth muscle cell and endothelium-derived agents modulate the muscle response. Several oxygen-sensing targets have been identified in smooth muscle, including potassium channels, Ca(2+) stores in the sarcoplasmic reticulum (SR) and the Ca(2+) sensitivity of the contractile proteins. The evidence for different oxygen-sensing mechanisms in pulmonary vessels is discussed.

Regional variation in P2 receptor expression in the rat pulmonary arterial circulation

The P2 receptors that mediate contraction of the rat isolated small (SPA, 200–500 μm i.d.) and large (LPA, 1–1.5 mM i.d.) intrapulmonary arteries were characterized. In endothelium‐denuded vessels the contractile order of potency was α,β‐methyleneATP (α,β‐meATP)>>UDP=UTP=ATP=2‐methylthioATP>ADP in the SPA and α,β‐meATP=UTPUDP>2‐methylthioATP, ATP>>ADP in the LPA. α,β‐meATP, 2‐methylthioATP and ATP had significantly greater effects in the SPA than the LPA (P<0.001), but there was no difference in the potency of UTP or UDP between the vessels. In the SPA, P2X1 receptor desensitisation by α,β‐meATP (100 μM) inhibited contractions to α,β‐meATP (10 nM–300 μM), but not those to UTP or UDP (100 nM–300 μM). In the LPA, prolonged exposure to α,β‐meATP (100 μM) did not desensitize P2X receptors. Pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), suramin and reactive blue 2 (RB2) (30–300 μM) inhibited contractions evoked by α,β‐meATP. UTP and UDP were potentiated by PPADS, unaffected by RB2 and inhibited, but not abolished by suramin. 1 and 3 mM suramin produced no further inhibition, indicating suramin‐resistant components in the responses to UTP and UDP. Thus, both P2X and P2Y receptors mediate contraction of rat large and small intrapulmonary arteries. P2Y agonist potency and sensitivity to antagonists were similar in small and large vessels, but P2X agonists were more potent in small arteries. This indicates differential expression of P2X, but not P2Y receptors along the pulmonary arterial tree.

Mechanisms of hydralazine induced vasodilation in rabbit aorta and pulmonary artery

The directly acting vasodilator hydralazine has been proposed to act at an intracellular site in vascular smooth muscle to inhibit Ca2+ release. This study investigated the mechanism of action of hydralazine on rabbit aorta and pulmonary artery by comparing its effects on the tension generated by intact and β‐escin permeabilized vessels and on the cytoplasmic Ca2+ concentration, membrane potential and K+ currents of isolated vascular smooth muscle cells. Hydralazine relaxed pulmonary artery and aorta with similar potency. It was equally effective at inhibiting phasic and tonic contractions evoked by phenylephrine in intact vessels and contractions evoked by inositol 1,4,5 trisphosphate (IP3) in permeabilized vessels. Hydralazine inhibited the contraction of permeabilized vessels and the increase in smooth muscle cell Ca2+ concentration evoked by caffeine with similar concentration dependence, but with lower potency than its effect on IP3 contractions. Hydralazine had no effect on the relationship between Ca2+ concentration and force generation in permeabilized vessels, but it slowed the rate at which maximal force was developed before, but not after, destroying sarcoplasmic reticulum function with the calcium ionophore, ionomycin. Hydralazine had no effect on membrane potential or the amplitudes of K+ currents recorded from isolated smooth muscle cells over the concentration range causing relaxation of intact vessels. The results suggest that the main action of hydralazine is to inhibit the IP3‐induced release of Ca2+ from the sarcoplasmic reticulum in vascular smooth muscle cells.