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D F Van Helden

Publications and source records attributed to D F Van Helden.

17 recordsLinked to original sources
PubMed1996

Endothelium-dependent modulation of pacemaking in lymphatic vessels of the guinea-pig mesentery.

1. Endothelial control of the rate of constrictions and the underlying pacemaker potentials has been studied in vitro in guinea-pig mesenteric lymphatic vessels. 2. ACh stimulated 60% of intraluminally perfused vessels to slow or abolish lymphatic constrictions. This action was inhibited by atropine and was likely to be due to the release of endothelium-derived nitric oxide (EDNO) as the effect was absent after endothelial lysis, mimicked by sodium nitroprusside (SNP), blocked by N omega-nitro L-arginine (NOLA) and partially inhibited by Methylene Blue (MB). 3. The remaining 40% of perfused vessels did not mechanically respond to ACh or SNP. In four of seven such vessels this appeared to be due to excessive perfusion-associated release of EDNO, as incubation with NOLA restored the response to SNP. 4. Application of NOLA or MB in perfused vessels significantly increased constriction frequency, further indicating perfusion-associated release of EDNO. 5. ACh induced a marked increase in endothelial [Ca2+]i of both mechanically responding and non-responding vessels. This ACh-induced increase could be repetitively induced when Ca2+ was present in the perfusate, but rapidly ran down when a Ca(2+)-free EGTA perfusate was used. 6. Intracellular recordings from the smooth muscle of non-perfused vessel segments demonstrated an ACh-induced hyperpolarization and decrease in membrane resistance, changes which were prevented by atropine, NOLA, MB and endothelial lysis and mimicked by SNP. 7. ACh directly reduced the size of the underlying pacemaker potentials termed spontaneous transient depolarizations (STDs). 8. NOLA and MB enhanced STDs in non-perfused vessel segments indicating an endogenous release of EDNO. 9. It is concluded that the lymphatic endothelium produces and releases EDNO endogenously, during perfusion or after stimulation with ACh, to decrease the efficacy of STDs to generate action potentials and resultant constrictions.

Acetylcholine↗
PubMed1996

The sigma receptor ligand, reduced haloperidol, induces apoptosis and increases intracellular-free calcium levels [Ca2+]i in colon and mammary adenocarcinoma cells.

The sigma receptor ligand reduced haloperidol (50 and 100 microM), potently inhibited cell proliferation, and induced apoptosis in WIDr colon and MCF-7 adenocarcinoma cell lines. Apoptosis was confirmed after drug treatment of the cells by the presence of nuclear fragmentation after staining of the cells with Hoechst 33258 and cellular DNA fragmentation ELISA and by condensation of the heterochromatin using transmission electron microscopy. However, internucleosomal DNA cleavage was not detected using gel electrophoresis. Reduced haloperidol (100 microM) increased the intracellular free calcium levels [Ca2+]i in both cell lines, which was independent of extracellular calcium, suggesting that the rise in [Ca2+]i was from intracellular stores and that an increase in [Ca2+]i may act as a "trigger" for apoptosis in these cell lines.

Adenocarcinoma↗
PubMed1996

Properties of spontaneous depolarizations in circular smooth muscle cells of rabbit urethra.

1. Intracellular microelectrode recordings were made from circular smooth muscle of rabbit urethra. 2. The smooth muscle of urethra was spontaneously active exhibiting large, regularly occurring depolarizations, termed slow waves (SWs), 1-3 s in duration, up to 40 mV in amplitude and generated every 3-15 s and small irregularly occurring events (or summations there of) termed spontaneous transient depolarizations (STDs) of < 1 s in duration. 3. The SWs and STDs were not sensitive to 10(-6) M atropine, 10(-6) M phentolamine, 10(-5) M guanethidine or 10(-6) M tetrodotoxin, indicating that they were myogenic in origin. 4. Application of 3 x 10(-6) M nifedipine or 5 x 10(-5) M GdCl3 did not inhibit the generation of SWs or STDs, indicating that activation of L-type Ca2+ channels and non-selective cation channels are not essential for their generation. However, the duration of SWs but not STDs was reduced by nifedipine, indicating L-type Ca2+ channels contribute to the plateau-like potential of SWs. 5. Application of low chloride solution (6.4 mM), niflumic acid (10(-5) - 10(-4) M) or 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS, 10(-4) -5 x 10(-4) M) inhibited the generation of SWs and STDs, suggesting an involvement of chloride channels. 6. Application of nominally Ca2+ free solution, 5 x 10(-5) M BAPTA-AM, 10(-5) M cyclopiazonic acid, 10(-2) M caffeine or 10(-3) M procaine inhibited the generation of SWs and STDs, indicating that Ca2+ released from intracellular stores was required for the generation of SWs and STDs. 7. Exogenously applied noradrenaline (10(-7) - 10(-5) M) increased the frequency of SWs through stimulation of alpha-adrenoceptors which was inhibited by sodium nitroprusside (SNP, 10(-4) M). SNP also reduced the frequency of SWs without altering the membrane potential, an effect mimicked by 8-bromocyclic GMP (10(-3) M) indicating that SNP acted by elevating the production of cyclic GMP. 8. It is concluded that smooth muscle cells of the rabbit urethra exhibit SWs and STDs which are likely to be induced by stimulation of Ca(2+)-activated chloride channels evoked by release of Ca2+ from intracellular stores.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1996

Modulation of frog skeletal muscle Ca2+ release channel gating by anion channel blockers.

Effects of niflumic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on frog skeletal muscle ryanodine receptors have been studied by incorporating sarcoplasmic reticulum vesicles into planar lipid bilayers. Niflumic acid increased the mean open probability (Po) at 10 microM and decreased Po at 100 microM with no change in open time constants, unitary conductance, and reversal potential. The Po was augmented by DIDS at 5-200 microM without affecting either unitary conductance or reversal potential. DIDS induced a new third open time constant, probably contributing to a long-lived open state. Channels modified by niflumic acid or DIDS still responded to Ca2+ release channel modulators. These results provide evidence that niflumic acid and DIDS modify the gating mechanism of ryanodine receptors without affecting binding sites to the modulators and the physical pathway of the conducting pore. p-Chloromercuriphenyl sulfonic acid (pCMPS) transiently increased the Po. The channel modified by DIDS responded to pCMPS, whereas that by ryanodine did not. The long open state of the channel induced by DIDS is produced by a quite different mechanism(s) from that by ryanodine. Contrary to cardiac ryanodine receptors, Po of skeletal muscle channels was independent of voltage after DIDS modification.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1996

Beta-adrenoceptor-mediated hyperpolarization in lymphatic smooth muscle of guinea pig mesentery.

Intracellular microelectrode recordings were performed to investigate the consequences of beta-adrenoceptor activation in smooth muscle of guinea pig mesenteric lymphatic vessels. Isoproterenol (Iso) hyperpolarized the membrane with an associated increase in membrane conductance and decreased the amplitude of spontaneous transient depolarizations. Iso effects were mimicked by forskolin (FSK), 3-isobutyl-1-methylxanthine, and two adenosine 3',5'-cyclic monophosphate (cAMP) derivatives. Iso- and FSK-induced hyperpolarizations were inhibited by H89, an inhibitor of cAMP-dependent protein kinase A, increased in K+-free solution, but were not affected by ouabain or by the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine. They were partially inhibited by 20 mM tetraethylammonium (approximately 40%) or by 2.5 mM 4-aminopyridine (approximately 55%). The-Iso-induced hyperpolarization was partially inhibited by iberiotoxin (20 nM) and charybdotoxin (40 nM), whereas the FSK-induced hyperpolarization was less affected. In cells where the Iso-induced hyperpolarization was decreased by 40 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, acetoxymethyl ester form, the FSK-induced hyperpolarization was little changed. Our results indicate that in guinea pig mesenteric lymphatic vessels, beta-adrenoceptor stimulation activates a protein kinase A-dependent K+ conductance, involving more than one channel type.

Animals↗
PubMed1995

Enhanced excitatory junction potentials in mesenteric arteries from spontaneously hypertensive rats.

Excitatory junction potentials (EJPs) were examined using intracellular recording techniques in mesenteric arteries isolated from 12- to 15-week-old spontaneously hypertensive (SHR), Wistar Kyoto (WKY) and Sprague Dawley (SD) rats. The amplitudes of EJPs evoked by single supramaximal stimuli were larger in arteries from SHRs (12.9 +/- 0.7 mV, n = 16) than in arteries from either WKYs (5.2 +/- 0.5 mV, n = 24) or SDs (8.6 +/- 0.8 mV, n = 15). The time constant of decay of EJPs did not differ significantly, suggesting that the passive electrical properties of the vascular smooth muscle are similar in the three rat strains. Spontaneous EJPs recorded in tissues from SHRs and WKYs had similar amplitude frequency distributions, suggesting that the quantal size is also similar between strains. In some arteries from SHRs, EJPs evoked by single stimuli triggered muscle action potentials (MAPs). Visible constriction only occurred following a MAP. In tissues from all three strains, summation of EJPs triggered MAPs. As EJPs are generated by the sympathetic co-transmitter adenosine 5'-triphosphate (ATP), the findings of the present study indicate that purinergic transmission is enhanced in mesenteric arteries from SHRs, probably as a result of an increase in quantal release. A consequence is that when nerves are activated SHR arteries more readily undergo constriction that is dependent on voltage-activated Ca2+ influx.

Action Potentials↗
PubMed1993

Ultrastructural analysis of sympathetic neuromuscular junctions on mesenteric veins of the guinea pig.

This study reports on the detailed ultrastructure of sympathetic postganglionic varicose axon terminals on mesenteric veins leading from the ileum of the guinea pig and in particular the structural arrangement of the varicosities with venous smooth muscle cells. The response to nerve stimulation in veins has a long time course and it has been suggested that this reflects a wide separation between the site of transmitter release and the receptors on the effector cell membrane. The aim of this study was to determine the distance between individual sympathetic varicosities and smooth muscle cells in mesenteric veins. Fluorescent histochemical preparations of the sympathetic innervation of the different branches of mesenteric veins indicate the branching network of varicose axons around the vessel to be relatively dense. Electron micrographs show the innervation to be confined to the adventitia close to the medio-adventitial border and to be predominantly catecholaminergic. A serial section ultrastructural analysis of the relationship of the varicosities with the outer smooth muscle cells showed that almost all (98%) of the exposed axon varicosities in the adventitia formed neuromuscular junctions. Three-dimensional reconstructions from serial sections of individual varicosities have shown that the junctions have structural specialisations identical to neuromuscular junctions described on arterial vessels and similar to those found at skeletal neuromuscular junctions. The density of neuromuscular junctions on the veins was found to be similar to that on the corresponding artery in the same animal. We suggest that in veins, noradrenaline is released focally at neuromuscular junctions.

Animals↗
PubMed1993

Edge detection at multiple locations using a 'radar' tracking algorithm as exemplified in isolated guinea-pig lymphatic vessels.

An algorithm based on the Kalman filter and used for tracking a target in a noisy environment has been applied to monitoring the position of moving boundaries as recorded by a video camera. The method, termed the Probabilistic Data Association Filter, is computationally efficient, allowing simultaneous monitoring at many positions along the boundary at video frame rates of 25 Hz. The present study uses the tracking procedure to investigate and correlate constrictions along lymphatic vessels. The procedure is well suited for real time tracking of visualised biological events including the simultaneous monitoring of constrictions in networks of blood vessels, the movement of cells or the trajectory of intracellular events monitored by intracellular fluorescent markers.

Algorithms↗
PubMed1993

Pacemaker potentials in lymphatic smooth muscle of the guinea-pig mesentery.

1. Intracellular microelectrode recordings have been made from lymphatic smooth muscle (SM) to investigate pacemaker mechanisms underlying lymphatic pumping. 2. The SM of small lymphangions or that of short segments, cut from lymphangions of any length, behaved similarly. Both preparations exhibited spontaneous transient depolarizations (STDs) and exhibited simplified electrical characteristics approximating those of a spherical cell. 3. STDs were found to underlie activation of action potentials and hence constrictions. 4. The level of STD activity correlated to the pumping activity of lymphangions, the SM from more active chambers exhibiting increased STD activity. 5. Lymphatic SM exhibited STDs with properties similar to the STDs of mesenteric veins. STDs appeared to be of myogenic origin as they were present despite denervation or substantial destruction of the endothelium. 6. Noradrenaline enhanced the size and frequency of STDs. 7. STD activity was abolished by chelation of cytosolic Ca2+. 8. It is proposed that STDs provide a mechanism for pacemaking in the lymphatic SM studied here. Furthermore, it is postulated that STDs are the consequence of Ca(2+)-dependent pulsatile release of an intracellular messenger, probably Ca2+ itself. This mechanism provides a novel means for pacemaking.

Action Potentials↗
PubMed1991

Spontaneous and noradrenaline-induced transient depolarizations in the smooth muscle of guinea-pig mesenteric vein.

1. Recordings of membrane current were made in the smooth muscle of short segments of mesenteric vein before or during stimulation with noradrenaline (NA). 2. Small veins (diameter less than 150 microns) when cut into short segments (of length less than 250 microns) had the passive electrical characteristics of short cables both before and during activation with NA. 3. Spontaneous transient depolarizations (STDs) or the underlying inward currents (STICs) were recorded in these preparations. STDs were of myogenic origin as they were not blocked by tetrodotoxin or antagonists to the alpha-adrenoreceptor and persisted after either denervation or disruption of the endothelium. 4. STDs had time courses similar to the underlying currents and were generally slow compared to the membrane time constant of the short segments. 5. STDs and the underlying currents showed large variability in frequency and amplitude both within and between short segments. Currents were typically less than 0.3 nA, were characteristic in shape, had half-durations normally in the range 0.1-0.7 s and reversed at about -25 mV. 6. STDs persisted, but at markedly reduced frequencies, after exposure (3-10 min) to a solution in which cobalt ions had been used to substitute for Ca2+. STDs were also substantially suppressed by exposure to low-chloride solution. 7. Caffeine induced excitatory and inhibitory conductances. An initial component of the caffeine-induced responses showed similar voltage dependence to STDs and was also suppressed by exposure to low-chloride solution. 8. NA, through activation of alpha-adrenoreceptors, caused a sustained depolarization or inward current (under voltage clamp) with considerable membrane potential or current noise often in the form of agonist-induced spontaneous transient depolarizations (ASTDs) or currents (ASTICs). There were marked increases in amplitude and frequency of ASTDs with increase in NA concentrations. 9. ASTDs appeared to be generated within the smooth muscle as they were activated in preparations which had been denervated or in which the endothelium had been disrupted. 10. Except for the pathway of activation, ASTDs were indistinguishable from STDs having half-durations in the same range (0.1-2 s with the majority less than 0.7 s). The underlying currents again showed large variation in amplitude (typically less than 0.3 nA; maximum recorded 0.9 nA). They reversed at about -25 mV, could still be elicited in cobalt solution (but at reduced intensity for long exposures to this low-Ca2+ solution) and were reduced by long term exposure to low-chloride solution.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Electrophysiology of neuromuscular transmission in guinea-pig mesenteric veins.

1. Neuromuscular transmission in the smooth muscle of mesenteric veins has been investigated by recording intracellular potential changes resulting from stimulation of the sympathetic nerves and comparing these potential changes with responses obtained by ionophoresis of noradrenaline. 2. Neural stimulation or exogenous noradrenaline acted similarly to cause two excitatory depolarizations, a slow response reported previously (Suzuki, 1981) and a separate fast depolarization. 3. The fast depolarization was distinct from the slow depolarizing response in that it had a different dependence on the level of stimulation, was readily desensitized and was more suppressed in low-chloride solution. 4. The fast but not the slow depolarization shared certain characteristics with constriction. The fast depolarization and constriction both increased with the intensity of stimulation; inactivation in both was dependent on the recovery interval between trains of stimuli and both were suppressed to a similar degree by antagonists to alpha-adrenoceptors. The fast depolarization was, however, not a prerequisite for constriction to occur. 5. The fast and slow depolarizations were activated after a long latency which had a high temperature coefficient consistent with the postulate that these responses are rate limited by intracellular biochemical reactions. 6. The fast depolarization was preferentially suppressed by prazosin, an antagonist to the alpha 1-adrenoceptor subtype. Suppression of the slow depolarization required relatively higher concentrations of antagonist, indicating that these responses were mediated by receptor interactions involving a different alpha-adrenoceptor subtype. 7. It is concluded that neuromuscular transmission in mesenteric veins occurs through activation of alpha-adrenoceptors. A number of responses result, including voltage-independent constriction and two distinct excitatory depolarizations which can lead to voltage-dependent constriction.

Action Potentials↗
PubMed1988

An alpha-adrenoceptor-mediated chloride conductance in mesenteric veins of the guinea-pig.

1. The ionic basis of the depolarizing responses resulting from ionophoresis of noradrenaline onto the smooth muscle of mesenteric veins has been investigated using electrically short segments of vessel. 2. Isolated cut segments of vein were effectively isopotential as assessed by the voltage response to a step change in current. The mean input resistance and time constant of the smooth muscle were 24 M omega and 131 ms respectively. 3. Data on the noradrenaline-induced slow depolarization indicated that it resulted from a decrease in conductance to potassium ions consistent with the finding of Suzuki (1981). 4. The fast noradrenaline-induced depolarization was found to have a reversal potential of about -22 mV. 5. Exposure to low-chloride solution caused greater than 90% suppression of this fast response with a 50% reduction occurring in less than 2 min. This suppression was not due to a negative shift in reversal potential. 6. The fast response underwent a large positive shift in reversal potential directly after changeover to low-chloride solution at times when any inactivation of the response was minimal. By contrast the fast response showed no evidence implicating either sodium or calcium as charge-carrying ions. 7. It is concluded the fast depolarization is carried by chloride ions.

Action Potentials↗
PubMed1984

Some properties of excitatory junction currents recorded from submucosal arterioles of guinea-pig ileum.

Excitatory junction potentials and excitatory junction currents have been recorded from short segments of arterioles taken from the submucosa of guinea-pig small intestine. Excitatory junction currents reached their peak amplitudes by 20 ms and then decayed, with a time course that could be described by a single exponential (mean time constant of decay approximately 50 ms). The null potential for excitatory junction currents obtained by extrapolation was near 0 mV. The time course of excitatory junction currents failed to show voltage sensitivity. Spontaneous excitatory junction currents had the same time course as evoked currents; the amplitudes of spontaneous currents were less than an order of magnitude different from that of evoked currents.

Action Potentials↗
PubMed1982

Fractionation of the asymmetry current in the squid giant axon into inactivating and non-inactivating components.

The operation of the voltage-sensitive sodium gating system in the nerve membrane involves conformational changes that are accompanied by small asymmetrical displacement currents. The asymmetry current may be divided into a component that is inactivated by positive voltage-clamp pulses, and recovers from inactivation with exactly the same time course as the sodium conductance, and one that is not inactivated. A method is described for recording the two components separately with the aid of an inactivating prepulse. They appear to have a marked difference in their rising phases, that of the non-inactivating component being just about as fast as the imposed step in membrane potential, while the inactivating component requires some tens of microseconds to reach its peak.

Animals↗
PubMed1982

The relationship between the inactivating fraction of the asymmetry current and gating of the sodium channel in the squid giant axon.

A quantitative comparison between the voltage dependence of the inactivating component of the asymmetrical charge transfer in the squid giant axon and that of the sodium conductance indicates that activation of the sodium system involves either three subunits operating in parallel or a three-step series mechanism. This is confirmed by an examination of the relative timing of the flow of asymmetry and ionic currents during the opening and closing of the sodium channels. In agreement with previous suggestions, inactivation is coupled sequentially to activation. The evidence appears to argue against a trimeric system with three wholly independent subunits and favours a monomeric system that undergoes a complex sequence of conformational changes.

Animals↗
PubMed1979

Cation permeation of the amphibian motor end-plate.

Measurements of acetylcholine-induced single-channel conductance and null potentials at the amphibian motor end-plate in solutions containing Na, K, Li and Cs ions (Gage & Van Helden, 1979; J. Physiol. (London) (in press) were analyzed in terms of three models. Two of these models, the "neutral" site channel model and the "charged" site channel model were developed to cater for three cations. Both were shown to be able to explain the dependence of single-channel conductance on membrane potential and gave the following sequences of equilibrium constants and mobilities. KLi/KNa/KK/KCs = 7:1.1:1:0.9 and UCs/UK/UNa/ULi = 1.4:1:0.58:0.13 at 8 degrees C. Similar sequences were obtained at 20 degrees C. Although the neutral model fitted the data for relative conductances in Li-, Cs- and Na-solutions slightly better than the charged model, experiments done in normal [NaCl] and [NaCl]/2 solutions could only be fitted by the neutral model. In contrast, the third model, the Constant Field Equation, was unable to fit the conductance data in any of the above situations. The data available suggests that permeation is through "long" neutral channels, lined with high field-strength negative polar groups and including one or possibly more high resistance barriers for anions.

Amphibians↗