Effect of chronic morphine treatment on alpha(2)-adrenoceptor mediated autoinhibition of transmitter release from sympathetic varicosities of the mouse vas deferens

1 The effect of chronic morphine treatment (CMT) on sympathetic innervation of the mouse vas deferens and on alpha (2)-adrenoceptor mediated autoinhibition has been examined using intracellular recording of excitatory junction potentials (EJPs) and histochemistry. 2 In chronically saline treated (CST) preparations. morphine (1 muM) and the alpha (2)-adrenoceptor agonist (clonidine, 1 muM) decreased the mean amplitude of EJPs evoked with 0.03 Hz stimulation by 81+/-8% (n=16) and 92+/-6% (n=7) respectively. In CMT preparations, morphine (1 muM) and clonidine (1 muM) decreased mean EJP amplitude by 68+/-8% (n = 7) and 79+/-8% (n = 7) respectively. 3 When stimulating the sympathetic axons at 0.03 Hz. the mean EJP amplitude recorded from smooth muscles acutely withdrawn from CMT was four times greater than for CST smooth muscles (40.7+/-3.8 mV, n = 7 compared with 9.9+/-0.3 mV, n = 7). 4 Part of the increase in mean EJP amplitude following CMT was produced by a 31% increase in the density of sympathetic axons and varicosities innervating the smooth muscle. 5 Results from the present study indicate that the effectiveness of alpha (2)-adrenocrptor mediated autoinhibition is only slightly reduced in CMT preparations. Most of the cross tolerance which develops between morphine, clonidine and alpha (2)-adrenoceptor mediated autoinhibition occurs as a consequence of increased efficacy of neuromuscular transmission which is produced by an increase in the probability of transmitter release and an increase in the density of sympathetic innervation.

Pre- and postsynaptic actions of ATP on neurotransmission in rat submandibular ganglia

The pre- and postsynaptic actions of exogenously applied ATP were investigated in intact and dissociated parasympathetic neurotics of rat submandibular ganglia. Nerve-evoked excitatory postsynaptic potentials (EPSPs) were not inhibited by the purinergic receptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2 ' ,4 ' -disulphonic acid (PPADS), or the desensitising agonist, alpha,beta -methylene ATP. In contrast. EPSPs were abolished by the nicotinic acetylcholine receptor antagonists, hexamethonium and mecamylamine. Focal application of ATP (100 muM) had no effect on membrane potential of the postsynaptic neurone or on the amplitude of spontaneous EPSPs. Taken together, these results suggest the absence of functional purinergic (P2) receptors on the postganglionic neurone in situ. In contrast, focally applied ATP (100 muM) reversibly inhibited nerve-evoked EPSPs. Similarly, bath application of the non-hydrolysable analogue of ATP, ATP gammaS, reversibly depressed EPSPs amplitude, The inhibitory effects of ATP and ATP gammaS on nerve-evoked transmitter release were antagonised by bath application of either PPADS or suramin, suggesting ATP activates a presynaptic P2 purinoceptor to inhibit acetylcholine release from preganglionic nerves in the submandibular ganglia. In acutely dissociated postganglionic neurotics from rat submandibular ganglia. focal application of ATP (100 LM) evoked an inward current and subsequent excitatory response and action potential firing, which was reversibly inhibited by PPADS (10 muM). The expression of P2X purinoceptors in wholemount and dissociated submandibular ganglion neurones was examined using polyclonal antibodies raised against the extracellular domain of six P2X purinoceptor subtypes (P2X(1-6)). In intact wholemount preparations, only the P2X(5) purinoceptor subtype was found to be expressed in the submandibular ganglion neurones and no P2X immunoreactivity was detected in the nerve fibres innervating the ganglion. Surprisingly, in dissociated submandibular ganglion neurones, high levels of P2X(2) and P2X(4) purinoceptors immunoreactivity were found on the cell surface. This increase in expression of P2X(2) and P2X(4) purinoceptors in dissociated submandibular neurones could explain the increased responsiveness of the neurotics to exogenous ATP. We conclude that disruption of ganglionic transmission in vivo by either nerve damage or synaptic blockade may up-regulate P2X expression or availability and alter neuronal excitability. (C) 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Large-conductance calcium-activated potassium channels in neonatal rat intracardiac ganglion neurons

The properties of single Ca2+-activated K+ (BK) channels in neonatal rat intracardiac neurons were investigated using the patch-clamp recording technique. In symmetrical 140 mM K+, the single-channel slope conductance was linear in the voltage range -60/+60 mV. and was 207+/-19 pS. Na+ ions were not measurably permeant through the open channel. Channel activity increased with the cytoplasmic free Ca2+ concentration ([Ca2+],) with a Hill plot giving a half-saturating [Ca2+] (K-0.5) of 1.35 muM and slope of congruent to3. The BK channel was inhibited reversibly by external tetraethylammonium (TEA) ions, charybdotoxin, and quinine and was resistant to block by 4-aminopyridine and apamin. Ionomycin (1-10 muM) increased BK channel activity in the cell-attached recording configuration. The resting activity was consistent with a [Ca2+](i)

Vascular endothelial growth factor-B-deficient mice show impaired development of hypoxic pulmonary hypertension

To test the hypothesis that Vegf-B contributes to the pulmonary vascular remodelling, and the associated pulmonary hypertension, induced by exposure of mice to chronic hypoxia. Methods: Right ventricular systolic pressure, the ratio of right ventricle/[left ventricle+septum] (RV/[LV+S]) and the thickness of the media (relative to vessel diameter) of intralobar pulmonary arteries (o.d. 50-150 and 151-420 mum) were determined in Vegfb knockout mice (Vegfb(-/-); n=17) and corresponding wild-type mice (Vegfb(+/+); n=17) exposed to chronic hypoxia (10% oxygen) or housed in room air (normoxia) for 4 weeks. Results: In Vegfb(+/+) mice hypoxia caused (i) pulmonary hypertension (a 70% increase in right ventricular systolic pressure compared with normoxic Vegfb(+/+) mice; P

Set-valued Markov chains and negative semitrajectories of discretized dynamical systems

Computer simulation of dynamical systems involves a phase space which is the finite set of machine arithmetic. Rounding state values of the continuous system to this grid yields a spatially discrete dynamical system, often with different dynamical behaviour. Discretization of an invertible smooth system gives a system with set-valued negative semitrajectories. As the grid is refined, asymptotic behaviour of the semitrajectories follows probabilistic laws which correspond to a set-valued Markov chain, whose transition probabilities can be explicitly calculated. The results are illustrated for two-dimensional dynamical systems obtained by discretization of fractional linear transformations of the unit disc in the complex plane.

Spatial structure and habitat variation in a grasshopper hybrid zone

A hybrid zone between the grasshoppers Chorthippus brunneus and C. jacobsi (Orthoptera: Acrididae) in northern Spain has been analyzed for variation in morphology and ecology. These species are readily distinguished by the number of stridulatory pegs on the hind femur. Both sexes are fully winged and inhabit disturbed habitats throughout the study area. We develop a maximum-likelihood approach to fitting a two-dimensional cline to geographical variation in quantitative traits and for estimating associations of population mean with local habitat. This method reveals a cline in peg number approximately 30 km south of the Picos de Europa Mountains that shows substantial deviations in population mean compared with the expectations of simple tension zone models. The inclusion of variation in local vegetation in the model explains a significant proportion of the residual variation in peg number, indicating that habitat-genotype associations contribute to the observed spatial pattern. However, this association is weak, and a number of populations continue to show strong deviations in mean even after habitat is included in the final model. These outliers may be the result of long-distance colonization of sites distant from the cline center or may be due to a patchy pattern of initial contact during postglacial expansion. As well as contrasting with the smooth hybrid zones described for Chorthippus parallelus, this situation also contrasts with the mosaic hybrid zones observed in Gryllus crickets and in parts of the hybrid zone between Bombina toad species, where habitat-genotype associations account for substantial amounts of among-site variation.

A monolithic smoothing-gap algorithm for contact-impact based on the signed distance function

A new algorithm has been developed for smoothing the surfaces in finite element formulations of contact-impact. A key feature of this method is that the smoothing is done implicitly by constructing smooth signed distance functions for the bodies. These functions are then employed for the computation of the gap and other variables needed for implementation of contact-impact. The smoothed signed distance functions are constructed by a moving least-squares approximation with a polynomial basis. Results show that when nodes are placed on a surface, the surface can be reproduced with an error of about one per cent or less with either a quadratic or a linear basis. With a quadratic basis, the method exactly reproduces a circle or a sphere even for coarse meshes. Results are presented for contact problems involving the contact of circular bodies. Copyright (C) 2002 John Wiley Sons, Ltd.

Branching processes and computational collapse of discretized unimodal mappings

In computer simulations of smooth dynamical systems, the original phase space is replaced by machine arithmetic, which is a finite set. The resulting spatially discretized dynamical systems do not inherit all functional properties of the original systems, such as surjectivity and existence of absolutely continuous invariant measures. This can lead to computational collapse to fixed points or short cycles. The paper studies loss of such properties in spatial discretizations of dynamical systems induced by unimodal mappings of the unit interval. The problem reduces to studying set-valued negative semitrajectories of the discretized system. As the grid is refined, the asymptotic behavior of the cardinality structure of the semitrajectories follows probabilistic laws corresponding to a branching process. The transition probabilities of this process are explicitly calculated. These results are illustrated by the example of the discretized logistic mapping.

Macrophages, myofibroblasts and neointimal hyperplasia after coronary artery injury and repair

Macrophages participate in the restenosis process through the release of cytokines, metalloproteinases and growth factors. Studies of peritoneal granulation tissue suggest that macrophages may be precursors of myofibroblasts. This study examined the contribution of monocyte/macrophage lineage cells to neointimal cellular mass in a porcine model of thermal vascular injury. Thermal coronary artery injury caused medial smooth muscle cell necrosis and transformation of the media into an extracellular matrix barrier. The neointimal hyperplasia that developed over the injury sites was evaluated by light microscopy, electron microscopy and immunohistochemistry. At day 3, blood monocytes were adhered to the vessel wall and infiltrated the fibrotic media. At day 14, 42 +/- 3.9% of neointimal cells had a monocytic nuclear morphology and expressed macrophage-specific antigen SWC3 (identified by monoclonal antibody DH59B). Moreover, 9.2+/-1.8% of neointimal cells co-expressed SWC3 and alpha-smooth muscle actin and had ultrastructural characteristics intermediate between macrophages and myofibroblasts. At day 28, 10.5 +/- 3.5%, of cells expressed SWC3 and 5.2+/-1.8% of cells co-expressed SWC3 and alpha-smooth muscle actin. This study indicates that hematopoietic cells of monocyte/macrophage lineage abundantly populate the neointima in the process of lesion formation and may be precursors of neointimal myofibroblasts after thermal vascular injury. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Basal nonselective cation permeability in rat cardiac microvascular endothelial cells

The presence of a basal nonselective cation permeability was mainly investigated in primary cultures of rat cardiac microvascular endothelial cells (CMEC) by applying both the patch-clamp technique and Fura-2 microfluorimetry. With low EGTA in the pipette solution, the resting membrane potential of CMEC was -21.2 +/- 1.1 mV, and a Ca2+-activated Cl- conductance was present. When the intracellular Ca2+ was buffered with high EGTA, the membrane potential decreased to 5.5 +/- 1.2 mV. In this condition, full or partial substitution of external Na+ by NMDG(+) proportionally reduced the inward component of the basal I-V relationship. This current was dependent on extracellular monovalent cations with a permeability sequence of K+ > Cs+ > Na+ > Li+ and was inhibited by Ca2+, La3+, Gd3+, and amiloride. The K+/Na+ permeability ratio, determined using the Goldman-Hodgkin-Katz equation, was 2.01. The outward component of the basal I-V relationship was reduced when intracellular K+ was replaced by NMDG(+), but was not sensitive to substitution by Cs+. Finally, microfluorimetric experiments indicated the existence of a basal Ca2+ entry pathway, inhibited by La3+ and Gd3+. The basal nonselective cation permeability in CMEC could be involved both in the control of myocardial ionic homeostasis, according to the model of the blood-heart barrier, and in the modulation of Ca2+ -dependent processes. (C) 2002 Elsevier Science (USA).

Inhibition of rat platelet aggregation by the diazeniumdiolate nitric oxide donor MAHMA NONOate

1 Inhibition of rat platelet aggregation by the nitric oxide (NO) donor MAHMA NONOate (Z-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-l-ium-1,2-diolate) was investigated. The aims were to compare its anti-aggregatory effect with vasorelaxation, to determine the effects of the soluble guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-ajquinoxalin-1-one), and to investigate the possible role of activation of sarco-encloplasmic reticulum calcium-ATPase (SERCA), independent of soluble guanylate cyclase, using thapsigargin. 2 MAHMA NONOate concentration-dependently inhibited sub-maximal aggregation responses to collagen (2 - 10 mug ml(-1)) and adenosine diphosphate (ADP; 2 mum) in platelet rich plasma. It was (i) more effective at inhibiting aggregation induced by collagen than by ADP, and (ii) less potent at inhibiting platelet aggregation than relaxing rat pulmonary artery. 3 ODQ (10 mum) caused only a small shift (approximately half a log unit) in the concentration-response curve to MAHMA NONOate irrespective of the aggregating agent. 4 The NO-independent activator of soluble guanylate cyclase, YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzy] indazole; 1 - 100 mum), did not inhibit aggregation. The cGMP analogue, 8-pCPT-cGMP (8-(4-chlorophenylthio)guanosine 3'5' cyclic monophosphate; 0.1 - 1 mm), caused minimal inhibition. 5 On collagen-aggregated platelets responses to MAHMA NONOate (ODQ 10 PM present) were abolished by thapsigargin (200 nm). On ADP-aggregated platelets thapsigargin caused partial inhibition. 6 Results with S-nitrosoglutathione (GSNO) resembled those with MAHMA NONOate. Glyceryl trinitrate and sodium nitroprusside were poor inhibitors of aggregation. 7 Thus inhibition of rat platelet aggregation by MAHMA NONOate (like GSNO) is largely ODQ-resistant and, by implication, independent of soluble guanylate cyclase. A likely mechanism of inhibition is activation of SERCA.

The current status of laser applications in dentistry

A range of lasers. is now available for use in dentistry. This paper summarizes key current and emerging applications, for lasers in clinical practice. A major diagnostic application of low power lasers is the detection of caries, using fluorescence elicited from hydroxyapatite or from bacterial by-products. Laser fluorescence is an effective method for detecting and quantifying incipient occlusal and cervical,carious lesions, and with further refinement could be used in the, same manner for proximal lesions. Photoactivated dye techniques have been developed which use low power lasers to elicit a photochemical reaction, Photoactivated dye techniques' can be used to disinfect root canals, periodontal pockets, cavity preparations and sites of peri-implantitis. Using similar principles, more powerful lasers tan be used for photodynamic therapy in the treatment of malignancies of the oral mucosa. Laser-driven photochemical reactions can also be used for tooth whitening. In combination with fluoride, laser irradiation can improve the resistance of tooth structure to demineralization, and this application is of particular benefit for susceptible sites in high caries risk patients. Laser technology for caries' removal, cavity preparation and soft tissue surgery is at a high state of refinement, having had several decades of development up to the present time. Used in conjunction with or as a replacement for traditional methods, it is expected that specific laser technologies will become an essential component of contemporary dental practice over the next decade.

Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane

The mechanisms involved in angiotensin II type 1 receptor (AT(1)-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT(1)-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT(1)-R in caveolae, AT(1)-R. caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT(1)-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT(1)-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT(1)-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT(1)-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT(1)-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT(1)-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT(1)-R through the exocytic pathway, but this does not result in AT(1)-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT(1)-R.

5-Hydroxytryptamine and platelets: uptake and aggregation in hypoxic pulmonary hypertensive rats

Pulmonary hypertension is associated with various alterations in 5-hydroxytryptamine (5-HT) physiology. In this study in platelets from hypoxic pulmonary hypertensive rats (10% O-2; 1 week) and normoxic rats (room air), (i) initial rates of specific [H-3]5-HT uptake were measured and (ii) potentiation of collagen- and ADP-induced aggregation by 5-HT was quantified. The platelet count was almost halved in hypoxic rats. In uptake experiments, there was a decrease in 5-HT uptake in platelets from hypoxic compared with normoxic rats, due to a 36% reduction in the maximal initial rate of uptake. The aggregation experiments showed that 5-HT (1-100 muM) increased the magnitude of responses to collagen and the duration of responses to ADP, but there was no difference between hypoxic and normoxic rats. Abnormalities in platelet function may conceivably lead to increases in plasma 5-HT levels in hypoxic pulmonary hypertension, but are unlikely to aggravate pulmonary thromboembolism. (C) 2002 Elsevier Science B.V. All rights reserved.

Potentiation of 5-hydroxytryptamine (5-HT) responses by a 5-HT uptake inhibitor in pulmonary and systemic vessels: effects of exposing rats to hypoxia

The aim was to determine whether uptake of 5-hydroxytryptamine (5-HT) by the 5-HT transporter (SERT) modulates contractile responses to 5-HT in rat pulmonary arteries and whether this modulation is altered by exposure of rats to chronic hypoxia (10% oxygen; 8 h/day; 5 days). The effects of the SERT inhibitor, citalopram (100 nM), on contractions to 5-HT were determined in isolated ring preparations of pulmonary artery (intralobar and main) and compared with data obtained in systemic arteries. In intralobar pulmonary arteries citalopram produced a potentiation (viz. an increase in potency, pEC(50)) of 5-HT. The potentiation was endothelium-dependent in preparations from normoxic rats but endothelium-independent in preparations from hypoxic rats. In main pulmonary artery endothelium-independent potentiation was seen in preparations from hypoxic rats but no potentiation occurred in preparations from normoxic rats. In systemic arteries, citalopram caused endothelium-independent potentiation in aorta but no potentiation in mesenteric arteries; there were no differences between hypoxic and normoxic rats. It is concluded that SERT can influence the concentration of 5-HT in the vicinity of the vasoconstrictor receptors in pulmonary arteries. The data suggest that in pulmonary arteries from hypoxic rats, unlike normoxic rats, the SERT responsible for this effect is not in the endothelium and, hence, is probably in the smooth muscle. The data are compatible with reports that, in the pulmonary circulation, hypoxia induces/up-regulates SERT, and hence increases 5-HT uptake, in vascular smooth muscle. The findings may have implications in relation to the suggested use of SERT inhibitors in the treatment of pulmonary hypertension.