Dirk F. van Helden

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 × 10−6 m nifedipine or 5 × 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 × 10−4 m) inhibited the generation of SWs and STDs, suggesting an involvement of chloride channels. 6 . Application of nominally Ca2+ free solution, 5 × 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 α‐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 Ca2+‐activated chloride channels evoked by release of Ca2+ from intracellular stores.

Epidermal Growth Factor Induces Tyrosine Phosphorylation, Membrane Insertion, and Activation of Transient Receptor Potential Channel 4

Various members of the canonical family of transient receptor potential channels (TRPCs) exhibit increased cation influx following receptor stimulation or Ca2+ store depletion. Tyrosine phosphorylation of TRP family members also results in increased channel activity; however, the link between the two events is unclear. We report that two tyrosine residues in the C terminus of human TRPC4 (hTRPC4), Tyr-959 and Tyr-972, are phosphorylated following epidermal growth factor (EGF) receptor stimulation of COS-7 cells. This phosphorylation was mediated by Src family tyrosine kinases (STKs), with Fyn appearing to be the dominant kinase. In addition, EGF receptor stimulation induced the exocytotic insertion of hTRPC4 into the plasma membrane dependent on the activity of STKs and was accompanied by a phosphorylation-dependent increase in the association of hTRPC4 with Na+/H+ exchanger regulatory factor. Furthermore, this translocation and association was defective upon mutation of Tyr-959 and Tyr-972 to phenylalanine. Significantly, inhibition of STKs was concomitant with a reduction in Ca2+ influx in both native COS-7 cells and hTRPC4-expressing HEK293 cells, with cells expressing the Y959F/Y972F mutant exhibiting a reduced EGF response. These findings represent the first demonstration of a mechanism for phosphorylation to modulate TRPC channel function.

Ca2+ phase waves: a basis for cellular pacemaking and long‐range synchronicity in the guinea‐pig gastric pylorus

Ca2+ imaging and multiple microelectrode recording procedures were used to investigate a slow wave‐like electrical rhythmicity in single bundle strips from the circular muscle layer of the guinea‐pig gastric pylorus. The ‘slow waves’ (SWs) consisted of a pacemaker and regenerative component, with both potentials composed of more elementary events variously termed spontaneous transient depolarizations (STDs) or unitary potentials. STDs and SW pacemaker and regenerative potentials exhibited associated local and distributed Ca2+ transients, respectively. Ca2+ transients were often larger in cellular regions that exhibited higher basal Ca2+ indicator‐associated fluorescence, typical of regions likely to contain intramuscular interstitial cells of Cajal (ICCIM). The emergence of rhythmicity arose through entrainment of STDs resulting in pacemaker Ca2+ transients and potentials, events that exhibited considerable spatial synchronicity. Application of ACh to strips exhibiting weak rhythmicity caused marked enhancement of SW synchronicity. SWs and underlying Ca2+ increases exhibited very high ‘apparent conduction velocities’ (‘CVs’) orders of magnitude greater than for sequentially conducting Ca2+ waves. Central interruption of either intercellular connectivity or inositol 1,4,5‐trisphosphate receptor (IP3R)‐mediated store Ca2+ release in strips caused SWs at the two ends to run independently of each other, consistent with a coupled oscillator‐based mechanism. Central inhibition of stores required much wider regions of blockade than inhibition of connectivity indicating that stores were voltage‐coupled. Simulations, made using a conventional store array model but now including depolarization coupled to IP3R‐mediated Ca2+ release, predicted the experimental findings. The linkage between membrane voltage and Ca2+ release provides a means for stores to interact as strongly coupled oscillators, resulting in the emergence of Ca2+ phase waves and associated pacemaker potentials. This distributed pacemaker triggers regenerative Ca2+ release and resultant SWs.

Generation and propagation of gastric slow waves

1. Mechanisms underlying the generation and propagation of gastrointestinal slow wave depolarizations have long been controversial. The present review aims to collate present knowledge on this subject with specific reference to slow waves in gastric smooth muscle.

Spontaneous transient depolarizations in lymphatic vessels of the guinea pig mesentery: pharmacology and implication for spontaneous contractility

Guinea pig mesenteric lymphatic vessels exhibit rhythmic constrictions induced by action potential (AP)-like spikes and initiated by entrainment of spontaneous transient depolarizations (STDs). To characterize STDs and the signaling mechanisms responsible for their occurrence, we used intracellular microelectrodes, Ca2+ imaging, and pharmacological agents. In our investigation of the role of intracellular Ca2+ released from Ca2+ stores, we observed that intracellular Ca2+ transients accompanied some STDs, although there were many exceptions where Ca2+ transients occurred without accompanying STDs. STD frequency and amplitude were markedly affected by activators/inhibitors of inositol 1,4,5-trisphosphate receptors (IP3Rs) but not by treatments known to alter Ca2+ release via ryanodine receptors. A role for Ca2+-activated Cl(-) (Cl(Ca)) channels was indicated, as STDs were dependent on the Cl(-) but not Na+ concentration of the superfusing solution and were inhibited by the Cl(Ca) channel blockers niflumic acid (NFA), anthracene 9-carboxylic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid but not by the volume-regulated Cl(-) blocker DIDS. Increases in STD frequency and amplitude induced by agonist stimulation were also inhibited by NFA. Nifedipine, the hyperpolarization-activated inward current blocker ZD-7288, and the nonselective cation/store-operated channel blockers SKF-96365, Gd3+, and Ni2+ had no or marginal effects on STD activity. However, nifedipine, 2-aminoethoxydiphenyl borate, NFA, SKF-96365, Gd3+, and Ni2+ altered the occurrence of spontaneous APs. Our findings support a role for Ca2+ release through IP3Rs and a resultant opening of Cl(Ca) channels in STD generation and confirm the importance of these events in the initiation of lymphatic spontaneous APs and subsequent contractions. The abolition of spontaneous APs by blockers of other excitatory ion channels suggests a contribution of these conductances to lymphatic pacemaking.

A Theoretical Model of Slow Wave Regulation Using Voltage-Dependent Synthesis of Inositol 1,4,5-Trisphosphate

A qualitative mathematical model is presented that examines membrane potential feedback on synthesis of inositol 1,4,5-trisphosphate (IP(3)), and its role in generation and modulation of slow waves. Previous experimental studies indicate that slow waves show voltage dependence, and this is likely to result through membrane potential modulation of IP(3). It is proposed that the observed response of the tissue to current pulse, pulse train, and maintained current injection can be explained by changes in IP(3), modulated through a voltage-IP(3) feedback loop. Differences underlying the tissue responses to current injections of opposite polarities are shown to be due to the sequence of events following such currents. Results from this model are consistent with experimental findings and provide further understanding of these experimental observations. Specifically, we find that membrane potential can induce, abolish, and modulate slow wave frequency by altering the excitability of the tissue through the voltage-IP(3) feedback loop.

Subpopulations of sympathetic neurones differ in their sensitivity to nerve growth factor antiserum.

Sympathetic postganglionic nerve fibres supplying mesenteric arteries and intrinsic ileal neurones differ in their characteristics of regeneration. Since the latter population of neurones occurs predominantly in prevertebral ganglia, which have been reported to be spared to some extent after treatment with antiserum to nerve growth factor (anti-NGF), we have investigated whether the two populations were differentially sensitive to anti-NGF. Newborn rats were treated daily for the first postnatal week with either anti-NGF or 154 mM NaCl solution. At 4 and 8 weeks of age, the presence of a functional sympathetic innervation to the mesenteric arteries and the gut was determined and correlated with the fluorescence histochemical demonstration of noradrenergic fibres. At both ages, stimulation of extrinsic sympathetic fibres caused an inhibition of gut motility, while the mesenteric arteries completely lacked a sympathetic innervation. Retrograde labelling of nerve cell bodies in control and antiserum treated rats confirmed that the sympathetic neurones supplying the ileal neurones were located in the prevertebral, superior mesenteric and coeliac ganglia and in the splanchnic ganglia lying along the greater splanchnic nerves. By interference from retrograde labelling in control animals, sympathetic neurones supplying the mesenteric arteries were present in all these ganglia, as well as in the thoracic and lumbar paravertebral sympathetic chains. The results suggest that two functionally distinct populations of sympathetic neurones, which overlap considerably in their distributions, are differentially sensitive to the immunological postnatal removal of NGF.

Evidence that the substance P-induced enhancement of pacemaking in lymphatics of the guinea-pig mesentery occurs through endothelial release of thromboxane A2.

1 In vitro studies were performed to examine the mechanisms underlying substance P‐induced enhancement of constriction rate in guinea‐pig mesenteric lymphatic vessels. 2 Substance P caused an endothelium‐dependent increase in lymphatic constriction frequency which was first significant at a concentration of 1 nM (115±3% of control, n=11) with 1 μM, the highest concentration tested, increasing the rate to 153±4% of control (n=9). 3 Repetitive 5 min applications of substance P (1 μM) caused tachyphylaxis with tissue responsiveness tending to decrease (by an average of 23%) and significantly decreasing (by 72%) for application at intervals of 30 and 10 min, respectively. 4 The competitive antagonist of tachykinin receptors, spantide (5 μM) and the specific NK1 receptor antagonist, WIN51708 (10 μM) both prevented the enhancement of constriction rate induced by 1 μM substance P. 5 Endothelial cells loaded with the Ca2+ sensing fluophore, fluo 3/AM did not display a detectable change in [Ca2+]i upon application of 1 μM substance P. 6 Inhibition of nitric oxide synthase by NG nitro‐L‐arginine (L‐NOARG; 100 μM) had no significant effect on the response induced by 1 μM substance P. 7 The enhancement of constriction rate induced by 1 μM substance P was prevented by the cyclo‐oxygenase inhibitor, indomethacin (3 μM), the thromboxane A2 synthase inhibitor, imidazole (50 μM), and the thromboxane A2 receptor antagonist, SQ29548 (0.3 μM). 8 The stable analogue of thromboxane A2, U46619 (0.1 μM) significantly increased the constriction rate of lymphangions with or without endothelium, an effect which was prevented by SQ29548 (0.3 μM). 9 Treatment with pertussis toxin (PTx; 100 ng ml−1) completely abolished the response to 1 μM substance P without inhibiting either the perfusion‐induced constriction or the U46619‐induced enhancement of constriction rate. 10 Application of the phospholipase A2 inhibitor, antiflammin‐1 (1 nM) prevented the enhancement of lymphatic pumping induced by substance P (1 μM), without inhibiting the response to either U46619 (0.1 μM) or acetylcholine (10 μM). 11 The data support the hypothesis that the substance P‐induced increase in pumping rate is mediated via the endothelium through NK1 receptors coupled by a PTx sensitive G‐protein to phospholipase A2 and resulting in generation of the arachidonic acid metabolite, thromboxane A2, this serving as the diffusible activator.

The Spectrin Cytoskeleton Influences the Surface Expression and Activation of Human Transient Receptor Potential Channel 4 Channels

Despite over a decade of research, only recently have the mechanisms governing transient receptor potential channel (TRPC) channel function begun to emerge, with an essential role for accessory proteins in this process. We previously identified a tyrosine phosphorylation event as critical in the plasma membrane translocation and activation of hTRPC4 channels following epidermal growth factor (EGF) receptor activation. To further characterize the signaling events underlying this process, a yeast-two hybrid screen was performed on the C terminus of hTRPC4. The intracellular C-terminal region from proline 686 to leucine 977 was used to screen a human brain cDNA library. Two members of the spectrin family, αII- and βV-spectrin, were identified as binding partners. The interaction of hTRPC4 with αII-spectrin and βV-spectrin was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation experiments. Deletion analysis identified amino acids 730-758 of hTRPC4 as critical for the interaction with this region located within a coiled-coil domain, juxtaposing the Ca2+/calmodulin- and IP3R-binding region (CIRB domain). This region is deleted in the proposed δhTRPC4 splice variant form, which failed to undergo both EGF-induced membrane insertion and activation, providing a genetic mechanism for regulating channel activity. We also demonstrate that the exocytotic insertion and activation of hTRPC4 following EGF application is accompanied by dissociation from αII-spectrin. Furthermore, depletion of αII-spectrin by small interference RNA reduces the basal surface expression of αhTRPC4 and prevents the enhanced membrane insertion in response to EGF application. Importantly, depletion of αII-spectrin did not affect the expression of the δ variant. Taken together, these results demonstrate that a direct interaction between hTRPC4 and the spectrin cytoskeleton is involved in the regulation of hTRPC4 surface expression and activation.

Evidence that the ATP-induced increase in vasomotion of guinea-pig mesenteric lymphatics involves an endothelium-dependent release of thromboxane A2

Experiments were made to investigate mechanisms by which adenosine 5′‐trisphosphate (ATP) enhanced vasomotion in mesenteric lymphatic vessels isolated from young guinea‐pigs. ATP (10−8–10−3 M) caused a concentration‐dependent increase of perfusion‐induced vasomotion with the endothelium mediating a fundamental role at low ATP concentrations (10−8–10−6 M). The response to 10−6 M ATP showed tachyphylaxis when applied at intervals of 10 min but not at intervals of 20 or 30 min. Suramin (10−4 M) or reactive blue 2 (3×10−5 M) but not PPADS (3×10−5 M) abolished the excitatory response to 10−6 M ATP confirming an involvement of P2 purinoceptors. The excitatory response to 10−6 M ATP was abolished by treatment with either pertussis toxin (100 ng ml−1), antiflammin‐1 (10−9 M), indomethacin (3×10−6 M) or SQ29548 (3×10−7 M), inhibitors of specific G proteins, phospholipase A2, cyclo‐oxygenase and thromboxane A2 receptors respectively. ATP simultaneously induced a suramin‐sensitive inhibitory response, which was normally masked by the excitatory response. ATP‐induced inhibition was mediated by endothelium‐derived nitric oxide (EDNO) as the response was abolished by NG‐nitro‐L‐arginine (L‐NOARG; 10−4 M), an inhibitor of nitric oxide synthase. We conclude that ATP modulates lymphatic vasomotion by endothelium‐dependent and endothelium‐independent mechanisms. One of these is a dominant excitation caused through endothelial P2 purinoceptors which because of an involvement of a pertussis toxin sensitive G‐protein may be of the P2Y receptor subtype. Their stimulation increases synthesis of phospholipase A2 and production of thromboxane A2, an arachidonic acid metabolite which acts as an endothelium‐derived excitatory factor.