Orline Bayguinov

β1-Subunits are required for regulation of coupling between Ca2+ transients and Ca2+-activated K+ (BK) channels by protein kinase C

Colonic myocytes have spontaneous, localized, Ins (1,4,5) trisphosphate (IP3) receptor-dependent Ca2+ transients that couple to the activation of Ca2+-dependent K+ channels and spontaneous transien...

Parallel pathways mediate inhibitory effects of vasoactive intestinal polypeptide and nitric oxide in canine fundus

The gastric adaptation reflex is activated by the release of non‐adrenergic, non‐cholinergic (NANC) inhibitory transmitters, including nitric oxide (NO) and vasoactive intestinal polypeptide (VIP). The role of NO in this reflex is not disputed, but some investigators suggest that NO synthesis is stimulated by VIP in post‐junctional cells or in nerve terminals. We investigated whether the effects of these transmitters are mediated by independent pathways in the canine gastric fundus. VIP and NO produced concentration‐dependent relaxation of the canine fundus. Nω‐nitro‐L‐arginine (L‐NNA) reduced relaxation induced by electrical field stimulation (EFS; 0.5–8 Hz), but had no effect on responses to exogenous VIP and sodium nitroprusside (SNP, 10 μM). Oxyhaemoglobin reduced relaxations produced by EFS and SNP. Oxyhaemoglobin also reduced relaxation responses to low concentrations of VIP (<10 nM), but these effects were non‐specific and mimicked by methaemoglobin which had no effect on nitrergic responses. A blocker of guanylyl cyclase, 1H‐[1,2,4]oxidiazolo [4,3,‐a]quinoxalin‐1‐one, (ODQ) inhibited responses to EFS, SNP and DETA/NONOate (an NO·donor), but had no effect on responses to VIP. cis‐N‐(2‐phenylcyclopentil)‐azacyclotridec‐1en‐2‐amine monohydrochloride (MDL 12,330A), a blocker of adenylyl cyclase, reduced responses to EFS, VIP and forskolin, but did not affect responses to SNP. Levels of cyclic GMP were enhanced by the NO donor S‐nitroso‐n‐acetylpenicillamine (SNAP) but were unaffected by VIP (1 μM). The increase in cyclic GMP in response to SNAP was blocked by ODQ. The results suggest that at least two transmitters, possibly NO and VIP, mediate relaxation responses in the canine fundus. NO and VIP mediate responses via cyclic GMP‐ and cyclic AMP‐dependent mechanisms, respectively. No evidence was found for a serial cascade in which VIP is coupled to NO‐dependent responses.

Small conductance Ca2+-activated K+ channels are regulated by Ca2+–calmodulin-dependent protein kinase II in murine colonic myocytes

1 Ca2+ regulates the activity of small conductance Ca2+‐activated K+ (SK) channels via calmodulin‐dependent binding. We investigated whether other forms of Ca2+‐dependent regulation might control the open probability of SK channels. 2 Under whole‐cell patch‐clamp conditions, spontaneous openings of SK channels can be resolved as charybdotoxin‐insensitive spontaneous transient outward currents (STOCs). The Ca2+‐calmodulin‐dependent (CaM) protein kinase II inhibitor KN‐93 reduced the occurrence of charybdotoxin‐insensitive STOCs. 3 The charybdotoxin‐insensitive STOCs are related to spontaneous, local release of Ca2+. KN‐93 did not affect spontaneous Ca2+‐release events. 4 KN‐93 and W‐7, a calmodulin inhibitor, decreased the open probability of SK channels in on‐cell patches but not in excised patches. 5 Application of autothiophosphorlated CaM kinase II to the cytoplasmic surface of excised patches increased the open probalibity of SK channels. Boiled CaM kinase II had no effect. 6 We conclude that CaM kinase II regulates SK channels in murine colonic myocytes. This mechanism provides a secondary means of regulation, increasing the impact of a given Ca2+ transient on SK channel open probability.

Movement based artifacts may contaminate extracellular electrical recordings from GI muscles.

Background  Electrical slow waves drive peristaltic contractions in the stomach and facilitate gastric emptying. In gastroparesis and other disorders associated with altered gastric emptying, motility defects have been related to altered slow wave frequency and disordered propagation. Experimental and clinical measurements of slow waves are made with extracellular or abdominal surface recording.

Substance P modulates localized calcium transients and membrane current responses in murine colonic myocytes

Neurokinins contribute to the neural regulation of gastrointestinal (GI) smooth muscles. We studied responses of murine colonic smooth muscle cells to substance P (SP) and NK1 and NK2 agonists using confocal microscopy and the patch clamp technique. Colonic myocytes generated localized Ca2+ transients that were coupled to spontaneous transient outward currents (STOCs). SP (10−10 M) increased Ca2+ transients and STOCs. Higher concentrations of SP (10−6 M) increased basal Ca2+ and inhibited Ca2+ transients and STOCs. Effects of SP were due to increased Ca2+ entry via L‐type Ca2+ channels, and were mediated by protein kinase C (PKC). Nifedipine (10−6 M) and the PKC inhibitor, GF 109203X (10−6 M) reduced L‐type Ca2+ current and blocked the effects of SP. SP responses depended upon parallel stimulation of NK1 and NK2 receptors. NK1 agonist ([Sar9,Met(O2)11]‐substance P; SSP) and NK2 agonists (neurokinin A (NKA) or GR‐64349) did not mimic the effects of SP alone, but NK1 and NK2 agonists were effective when added in combination (10−10–10−6 M). Consistent with this, either an NK1‐specific antagonist (GR‐82334; 10−7 M) or an NK2‐specific antagonist (MEN 10,627; 10−7 M) blocked responses to SP (10−6 M). Ryanodine (10−5 M) blocked the increase in Ca2+ transients and STOCs in response to SP (10−10 M). Our findings show that low concentrations of SP, via PKC‐dependent enhancement of L‐type Ca2+ current and recruitment of ryanodine receptors, stimulate Ca2+ transients. At higher concentrations of SP (10−6 M), basal Ca2+ increases and spontaneous Ca2+ transients and STOCs are inhibited.

Regulation of neural responses in the canine pyloric sphincter by opioids

1 Regulation of excitatory and inhibitory junction potentials (e.j.ps and i.j.ps) by opioid peptides was studied in isolated muscle strips from the pyloric sphincter of the dog. 2 Methionine enkephalin (MetEnk; 10−10 to 10−6 m) and [d‐Ala2, d‐Leu5] enkephalin (DADLE; 10−11 to 10−7 m), a δ‐specific opioid agonist, inhibited i.j.ps and e.j.ps recorded from cells in the myenteric and submucosal regions of the circular muscle layer. These compounds had no effect on resting potential or slow wave activity suggesting that the effects on junction potentials were not due to direct effects on smooth muscle cells. 3 MetEnk and DADLE caused similar effects on junction potentials in preparations in which the myenteric plexus was removed, suggesting that opioids inhibit pre‐junctional effects on nerve fibres within the muscularis externa. 4 Inhibition of junction potentials by MetEnk and DADLE was blocked by approximately the same extent by naloxone (10−6 m) and ICI 174,864 (10−6 m), a δ‐specific antagonist. 5 MetEnk and DADLE blocked a portion of the i.j.p. that was sensitive to arginine analogues; after treatment with Nω‐nitro‐l‐arginine methyl ester (l‐NAME, 10−4 m), MetEnk and DADLE had no further effect on i.j.ps. These data suggest that opioids regulate nitric oxide‐dependent neurotransmission. 6 Naloxone (10−6 m) alone had no effect on i.j.ps elicited by short trains of electrical field stimuli. 7 I.j.p. amplitude was reduced after a period of conditioning stimulation (2 min, 30 Hz, 30 V). Naloxone blocked the post‐stimulation inhibition. Repetitive stimulation at high frequencies (30 Hz) resulted in sustained hyperpolarization. Naloxone increased the amplitude of the hyperpolarization responses elicited by high frequency stimulation. 8 These results show that e.j.ps and i.j.ps in the canine pylorus are inhibited by opioids. A portion of the inhibitory effects appears to be mediated via δ receptors. 9 Although pyloric muscles are richly innervated by nerves containing opioid peptides, brief trains of stimuli do not appear to release concentrations of opioids that are effective in regulating junction potentials. Higher frequency stimulation (or longer durations of stimulation) appear to be necessary to release concentrations of opioids that are effective in modulating the amplitude of junction potentials.

Dissociation between electrical and mechanical responses to nitrergic stimulation in the canine gastric fundus

1 We examined the relationships between membrane potential, intracellular [Ca2+] ([Ca2+]i), and tension in muscles of the canine gastric fundus in response to nitrergic stimulation by NO donors and electrical field stimulation (EFS) of intrinsic enteric inhibitory neurons when adrenergic and cholinergic responses were blocked. 2 NO donors reduced [Ca2+]i and tension in a concentration‐dependent manner. A close relationship was noted between these parameters. 3 In terms of the [Ca2+]vs. force relationship, relaxation responses to EFS differed from responses to NO donors. EFS resulted in smaller decreases in [Ca2+]i to produce a given relaxation compared with responses to NO donors. Thus, muscles stimulated with EFS were less sensitive to [Ca2+]i than muscles stimulated with exogenous NO. 4 When membrane potential, [Ca2+]i and tension were monitored simultaneously in the same muscles, a temporal dissociation was noted between the electrical responses and changes in [Ca2+]i and tension. Brief electrical responses were associated with more sustained changes in [Ca2+]i and tension. 5 Further dissociation between electrical and mechanical effects was noted. Changes in [Ca2+]i and tension caused by sodium nitroprusside and EFS were blocked by arginine analogues and by oxyhaemoglobin, but electrical responses were unaffected. 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ), an inhibitor of soluble guanylyl cyclase, also blocked the effects of nitrergic stimulation on [Ca2+]i and tension, without affecting hyperpolarization. Thus, in the presence of continued hyperpolarization, the reductions in [Ca2+]i and tension caused by nitrergic stimulation were blocked. 6 Block of hyperpolarization in response to nitrergic stimulation with tetrapentylammonium chloride (TPEA) had relatively little effect on the [Ca2+]i and tension responses. Thus, hyperpolarization is not required for nitrergic effects on [Ca2+]i and tension. 7 In summary, reduction in [Ca2+]i and tension in response to nitrergic stimulation of the canine gastric fundus does not depend upon electrical hyperpolarization. Non‐electrical mechanisms such as enhanced uptake of Ca2+ by the sarcoplasmic reticulum or reduction in the Ca2+ sensitivity of the contractile apparatus may be the primary mechanisms mediating nitrergic responses in these muscles.

Response to Dr Nakayama letter

Dear Editor, We thank Dr. Nakayama for his acknowledgment that movement can be a significant artifact in extracellular recordings of biopotentials. In our article we showed that gastric movements are not slow monophasic events as described by Dr. Nakayama. We tracked small dots affixed to the gastric serosa to understand the movement trajectories that might influence an electrode against the surface. With each contractile cycle, the dots moved in a complex orbits, changing direction repeatedly. These tiny movements correlated with and created relatively large artifacts in electrical recordings and are likely to be the main source of the spiky electrical records, not Ca currents or action potentials. In fact, much of the gastric musculature does not experience Ca action potentials, even during stimulation by agonists, Our intracellular electrical recording from mice confirm these findings. We also used wortmannin, a myosin light chain kinase inhibitor, to suppress movement that does not affect currents flowing across cell membranes (resting membrane potentials and slow waves were unaffected by wortmannin). Wortmannin also blocked biopotentials, strengthening the conclusion that extracellular potentials recorded by surface electrodes are largely movement artifacts. Proper filtering is important, and we agree that filtering affects the waveforms recorded by extracellular techniques. We initially chose bandpass characteristics like those used by Professor William Lammers in his many studies of GI biopotentials. In contrast we also sampled between (0.3–100 Hz), and this did not affect our results or interpretations. The main signal one expects to record from stomach would result from rapid, large changes in membrane potential that occur during slow wave upstrokes (>1 V s when recording intracellularly from interstitial cells of Cajal (ICC); cells that generate and actively propagate slow waves). The upstroke mechanism is analogous to the Na current of cardiac action potentials. In ECGs the QRS (representing the action potential upstroke) is resolved with greatest amplitude and signal to noise ratio. Gastric slow waves are far slower in rise-time (about 100-fold) and far smaller in amplitude (about half) than cardiac action potentials, and in the bowel only ICC actively regenerate slow waves. So only a fraction of cells in the gut (<10%) are active current sources generating field potentials. Low pass filtering <1 Hz would attenuate fast changes in potential during slow waves, and this would attenuate the signals most likely to be resolved by extracellular recording. Our findings make it clear that careful suppression of movement is an essential control for experiments employing extracellular electrical recording.