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1.
Am J Physiol Cell Physiol ; 293(5): C1645-59, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17855773

ABSTRACT

Electrical slow waves determine the timing and force of peristaltic contractions in the stomach. Slow waves originate from a dominant pacemaker in the orad corpus and propagate actively around and down the stomach to the pylorus. The mechanism of slow-wave propagation is controversial. We tested whether Ca(2+) entry via a voltage-dependent, dihydropyridine-resistant Ca(2+) conductance is necessary for active propagation in canine gastric antral muscles. Muscle strips cut parallel to the circular muscle were studied with intracellular electrophysiological techniques using a partitioned-chamber apparatus. Slow-wave upstroke velocity and plateau amplitude decreased from the greater to the lesser curvature, and this corresponded to a decrease in the density of interstitial cells of Cajal in the lesser curvature. Slow-wave propagation velocity between electrodes impaling cells in two regions of muscle and slow-wave upstroke and plateau were measured in response to experimental conditions that reduce the driving force for Ca(2+) entry or block voltage-dependent Ca(2+) currents. Nicardipine (0.1-1 microM) did not affect slow-wave upstroke or propagation velocities. Upstroke velocity, amplitude, and propagation velocity were reduced in a concentration-dependent manner by Ni(2+) (1-100 microM), mibefradil (10-30 microM), and reduced extracellular Ca(2+) (0.5-1.5 mM). Depolarization (by 10-15 mM K(+)) or hyperpolarization (10 microM pinacidil) also reduced upstroke and propagation velocities. The higher concentrations (or lowest Ca(2+)) of these drugs and ionic conditions tested blocked slow-wave propagation. Treatment with cyclopiazonic acid to empty Ca(2+) stores did not affect propagation. These experiments show that voltage-dependent Ca(2+) entry is obligatory for the upstroke phase of slow waves and active propagation.


Subject(s)
Calcium Channels/metabolism , Calcium Signaling , Muscle, Smooth/metabolism , Myoelectric Complex, Migrating , Peristalsis , Pyloric Antrum/metabolism , Animals , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Calcium Channels/drug effects , Calcium Signaling/drug effects , Calcium-Transporting ATPases/antagonists & inhibitors , Calcium-Transporting ATPases/metabolism , Dogs , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , In Vitro Techniques , Indoles/pharmacology , Mibefradil/pharmacology , Muscle, Smooth/drug effects , Muscle, Smooth/enzymology , Myoelectric Complex, Migrating/drug effects , Nicardipine/pharmacology , Nickel/metabolism , Peristalsis/drug effects , Pinacidil/pharmacology , Potassium/metabolism , Potassium Channels/drug effects , Potassium Channels/metabolism , Pyloric Antrum/drug effects , Pyloric Antrum/enzymology , Stromal Cells/metabolism , Time Factors
2.
Am J Physiol Cell Physiol ; 291(2): C375-85, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16571863

ABSTRACT

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been suggested as participants in enteric inhibitory neural regulation of gastrointestinal motility. These peptides cause a variety of postjunctional responses including membrane hyperpolarization and inhibition of contraction. Neuropeptides released from enteric motor neurons can elicit responses by direct stimulation of smooth muscle cells as opposed to other transmitters that rely on synapses between motor nerve terminals and interstitial cells of Cajal. Therefore, we studied the responses of murine colonic smooth muscle cells to VIP and PACAP(1-38) with confocal microscopy and patch-clamp technique. Localized Ca2+ transients (Ca2+ puffs) were observed in colonic myocytes, and these events coupled to spontaneous transient outward currents (STOCs). VIP and PACAP increased Ca2+ transients and STOC frequency and amplitude. Application of dibutyryl cAMP had similar effects. The adenylyl cyclase blocker MDL-12,330A alone did not affect spontaneous Ca2+ puffs and STOCs but prevented responses to VIP. Disruption of A-kinase-anchoring protein (AKAP) associations by application of AKAP St-Ht31 inhibitory peptide had effects similar to those of MDL-12,330A. Inhibition of ryanodine receptor channels did not block spontaneous Ca2+ puffs and STOCs but prevented the effects of dibutyryl cAMP. These findings suggest that regulation of Ca2+ transients (which couple to activation of STOCs) may contribute to the inhibitory effects of VIP and PACAP. Regulation of Ca2+ transients by VIP and PACAP occurs via adenylyl cyclase, increased synthesis of cAMP, and PKA-dependent regulation of ryanodine receptor channels.


Subject(s)
Calcium Signaling/physiology , Calcium-Binding Proteins/metabolism , Colon/metabolism , Cyclic AMP/metabolism , Myocytes, Smooth Muscle/metabolism , Peptide Fragments/pharmacology , Pituitary Adenylate Cyclase-Activating Polypeptide/pharmacology , Vasoactive Intestinal Peptide/pharmacology , Animals , Calcium Signaling/drug effects , Calcium-Binding Proteins/genetics , Cells, Cultured , Colon/drug effects , Feedback/physiology , Female , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Myocytes, Smooth Muscle/drug effects , Signal Transduction/drug effects , Signal Transduction/physiology
3.
Am J Physiol Cell Physiol ; 285(5): C1270-80, 2003 Nov.
Article in English | MEDLINE | ID: mdl-12867357

ABSTRACT

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 transient outward currents (STOCs). We previously reported that the coupling strength between spontaneous Ca2+ transients and large conductance Ca2+ activated K+ (BK) channels is regulated by Ca2+ influx through nonselective cation channels and activation of protein kinase C (PKC). Here, we used confocal microscopy and the patch-clamp technique to further investigate the coupling between localized Ca2+ transients and STOCs in colonic myocytes from animals lacking the regulatory beta1-subunit of BK channels. Myocytes from beta1-knockout (beta1-/-) animals loaded with fluo 4 showed typical localized Ca2+ transients, but the STOCs coupled to these events were of abnormally low amplitude. Reduction in external Ca2+ or application of inhibitors of nonselective cation channels (SKF-96365) caused no significant change in the amplitude or frequency of STOCs. Likewise, an inhibitor of PKC, GF 109203X, had no significant effect on STOCs. Single-channel recording from BK channels showed that application of an activator (PMA) and an inhibitor (GF 109203X) of PKC did not affect BK channel openings in myocytes of beta1-/- mice. These data show that PKC-dependent regulation of coupling strength between Ca2+ transients and STOCs in colonic myocytes depends upon the interaction between alpha- and beta1-subunits.


Subject(s)
Calcium/metabolism , Potassium Channels, Calcium-Activated/physiology , Protein Kinase C/metabolism , Animals , Colon/cytology , Colon/enzymology , Colon/metabolism , Colon/physiology , Female , Large-Conductance Calcium-Activated Potassium Channels , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Muscle Cells/cytology , Muscle Cells/enzymology , Muscle Cells/metabolism , Muscle Cells/physiology , Potassium Channels, Calcium-Activated/deficiency , Potassium Channels, Calcium-Activated/genetics , Protein Subunits/deficiency , Protein Subunits/genetics , Protein Subunits/physiology
4.
Br J Pharmacol ; 138(7): 1233-43, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12711623

ABSTRACT

1. 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 NK(1) and NK(2) agonists using confocal microscopy and the patch clamp technique. 2. Colonic myocytes generated localized Ca(2+) transients that were coupled to spontaneous transient outward currents (STOCs). SP (10(-10) M) increased Ca(2+) transients and STOCs. Higher concentrations of SP (10(-6) M) increased basal Ca(2+) and inhibited Ca(2+) transients and STOCs. 3. Effects of SP were due to increased Ca(2+) entry via L-type Ca(2+) 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 Ca(2+) current and blocked the effects of SP. 4. SP responses depended upon parallel stimulation of NK(1) and NK(2) receptors. NK(1) agonist ([Sar(9),Met(O(2))(11)]-substance P; SSP) and NK(2) agonists (neurokinin A (NKA) or GR-64349) did not mimic the effects of SP alone, but NK(1) and NK(2) agonists were effective when added in combination (10(-10)-10(-6) M). Consistent with this, either an NK(1)-specific antagonist (GR-82334; 10(-7) M) or an NK(2)-specific antagonist (MEN 10,627; 10(-7) M) blocked responses to SP (10(-6) M). 5. Ryanodine (10(-5) M) blocked the increase in Ca(2+) transients and STOCs in response to SP (10(-10) M). 6. Our findings show that low concentrations of SP, via PKC-dependent enhancement of L-type Ca(2+) current and recruitment of ryanodine receptors, stimulate Ca(2+) transients. At higher concentrations of SP (10(-6) M), basal Ca(2+) increases and spontaneous Ca(2+) transients and STOCs are inhibited.


Subject(s)
Calcium Signaling/drug effects , Colon/cytology , Colon/drug effects , Electric Conductivity , Neurokinin A/analogs & derivatives , Physalaemin/analogs & derivatives , Substance P/pharmacology , Animals , Calcium Signaling/physiology , Colon/metabolism , Imidazoles/pharmacology , Indoles/pharmacology , Male , Maleimides/pharmacology , Membrane Potentials/drug effects , Mice , Mice, Inbred BALB C , Microscopy, Confocal , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Neurokinin A/pharmacology , Nicardipine/pharmacology , Patch-Clamp Techniques , Peptide Fragments/pharmacology , Peptides/pharmacology , Physalaemin/pharmacology , Receptors, Tachykinin/drug effects , Receptors, Tachykinin/physiology , Ryanodine/pharmacology , Ryanodine Receptor Calcium Release Channel/drug effects , Second Messenger Systems/drug effects , Second Messenger Systems/physiology , Substance P/analogs & derivatives
5.
Gastroenterology ; 122(5): 1442-54, 2002 May.
Article in English | MEDLINE | ID: mdl-11984529

ABSTRACT

BACKGROUND & AIMS: Cyclooxygenase enzymes (COX) generate intermediates in the prostaglandin (PG) cascade. COX-1 is constitutively expressed in many cells, and COX-2 is typically thought to be an inducible isoform. METHODS: We evaluated constitutive expression and function of COX-2 in murine gastric muscles. RESULTS: Immunohistochemistry showed COX-2-like immunoreactivity (COX-2-LI) in myenteric neurons. Half the neurons with COX-2-LI expressed nitric oxide synthase (NOS). COX-2-LI was not observed in smooth muscle cells. Interstitial cells of Cajal within muscle layers (IC-IM) expressed COX-2-LI, suggesting a novel role for IC-IM. Molecular studies verified expression of COX-2 in gastric muscles. Quantitative polymerase chain reaction (PCR) showed equal expression of COX-1 and COX-2 in the antrum. COX-2 was more abundant in fundus. Indomethacin and GR253035X, a COX-2 inhibitor, increased antral phasic contractions and potentiated responses to ACh. Indomethacin, but not GR253035X, increased contractions and potentiated responses in tissues of COX-2 knockout mice. Indomethacin and GR253035X reduced tone in the fundus. CONCLUSIONS: COX-2 is constitutively expressed by IC-IM and neurons in the stomach and at levels similar to COX-1. Prostanoids produced by COX-2 regulate mechanical activities of fundus and antral muscles.


Subject(s)
Isoenzymes/analysis , Prostaglandin-Endoperoxide Synthases/analysis , Stomach/enzymology , Acetylcholine/pharmacology , Animals , Cyclooxygenase 2 , Female , Immunohistochemistry , Indomethacin/pharmacology , Isoenzymes/genetics , Isoenzymes/physiology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Muscle Contraction/drug effects , Nitric Oxide Synthase/analysis , Nitroprusside/pharmacology , Prostaglandin-Endoperoxide Synthases/genetics , Prostaglandin-Endoperoxide Synthases/physiology , RNA, Messenger/analysis , Stomach/drug effects , Stomach/physiology
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