Biomedical subjects

K M Sanders

Publications and source records attributed to K M Sanders.

At least 145 records · Page 8Linked to original sources

PubMed1994

Relationship between nitric oxide and vasoactive intestinal polypeptide in enteric inhibitory neurotransmission.

Although considerable evidence suggests that NO serves as a neurotransmitter in gastrointestinal muscles, it is unlikely to be the only substance involved in enteric inhibitory neurotransmission. Vasoactive intestinal polypeptide (VIP) is known to be expressed by inhibitory motor neurons in the gut, and it appears to be co-localized with nitric oxide synthase (NOS) in a subpopulation of enteric neurons. These data suggest that NO and VIP may be parallel neurotransmitters. Others have suggested that VIP is the primary inhibitory transmitter, and it stimulates production of NO in smooth muscle cells. In this "serial cascade" model NO is a paracrine substance. We performed experiments on circular muscles and cells from the canine proximal colon to further test the idea that NO and VIP are parallel neurotransmitters and to determine the validity of the serial cascade model in these muscles. We found that NO-independent inhibitory effects were unmasked when excitatory and NO-dependent inhibitory responses were blocked. NO-independent inhibitory effects were reduced by alpha-chymotrypsin and blocked by tetrodotoxin. NOS- and VIP-like immunoreactivities were co-localized in enteric neurons and varicose fibers in the circular muscle layer. Similar to several other reports we found no evidence for a constitutive NOS in smooth muscle cells. Several aspects of the serial cascade model were not supported by our results: (i) the electrical and mechanical effects of VIP did not depend upon NO synthesis; (ii) VIP-induced changes in [Ca2+]i did not depend upon NO synthesis; and (iii) VIP did not cause the release of NO from canine colonic muscles. These results are consistent with the hypothesis that NO and VIP are co-transmitters, released in parallel from enteric inhibitory nerves.

Animals↗

PubMed1994

Leukemic arthritis.

Leukemic arthritis (LA) is an uncommon complication of both acute and chronic leukemias. LA occurs in 12% to 65% of childhood leukemia cases and 4% to 13% of adult leukemia cases. Involved joints usually are warm, swollen, and tender. This arthritis often is pauciarticular and preferentially involves large joints. LA occurs at any time during the course of leukemia and may be the presenting manifestation. Therefore, LA should be considered in the differential diagnosis of both adult and childhood rheumatic diseases. Establishing a diagnosis may be difficult and usually requires tissue biopsy. Newer immunochemical methods may help in the diagnosis of LA by demonstrating leukemic cells in synovial fluid. Therapy for the underlying leukemia is the treatment of choice. Decreasing joint pain often is the first sign of a clinical response to chemotherapy. A case report is presented and the literature on the subject is reviewed.

Arthritis↗

PubMed1994

Expression of nitric oxide synthase immunoreactivity by interstitial cells of the canine proximal colon.

A subpopulation of interstitial cells (ICs) are interposed between nerve terminals and smooth muscle cells in the gastrointestinal tract and may participate in neuromuscular transmission. These cells appear to be targets for NO released from enteric inhibitory nerves and respond to exogenous NO with: (i) an elevation in cGMP levels; (ii) an increase in intracellular Ca2+; (iii) and release of a diffusible substance that has tentatively been identified as NO. For the latter to be possible, ICs must express a constitutive isoform of NOS. This study characterized the expression of NOS-like immunoreactivity (NOS-LI) in ICs of the canine colon using 3 antibodies raised against the 2 known constitutive forms of NOS (i.e., neural (nNOS) and endothelial (eNOS) isoforms). Antibodies raised against cNOS and an antibody raised against rat cerebellar nNOS labeled ICs along the submucosal surface of the circular muscle layer (IC-SM), along the surface of septa that separate the circular muscle into fiber bundles (IC-SM), and in the myenteric region between the circular and longitudinal muscle layers (IC-MY). Another antibody raised against rat cerebellar nNOS failed to label ICs. Cultured IC-SM also expressed NOS-LI, suggesting that this feature of the IC phenotype survives culture conditions. Arteriolar endothelial cells in the canine colon were labeled with the same 2 antibodies that labeled ICs, suggesting there are significant structural similarities between NO synthases in ICs and endothelial cells. The data suggest that IC-SM and IC-MY express a constitutive form of NOS. Synthesis of NO by ICs may influence electrical rhythmicity and may serve to amplify and even propagate enteric inhibitory neurotransmission.

Amino Acid Oxidoreductases↗

PubMed1994

Cloning and characterization of a Kv1.5 delayed rectifier K+ channel from vascular and visceral smooth muscles.

We have cloned and characterized the expression of a Kv1.5 K+ channel (cKv1.5) from canine colonic smooth muscle. The amino acid sequence displayed a high level of identity to other K+ channels of the Kv1.5 class in the core region between transmembrane segments S1-S6; however, identity decreased to between 74 and 82% in the NH2 and COOH terminal segments, suggesting that cKv1.5 is a distinct isoform of the Kv1.5 class. Functional expression of cKv1.5 in oocytes demonstrated a channel highly selective for K+, which activates in a voltage-dependent manner on depolarization to membrane potentials positive to -40 mV. At room temperature the channel showed fast activation (time to half of peak current, 5.5 ms) and slow inactivation that was incomplete after 20-s depolarizations. Single channel analysis of the channel expressed in oocytes displayed a linear current-voltage curve and had a slope conductance of 9.8 +/- 1.1 pS. Northern blot analysis demonstrated differential expression of cKv1.5 in smooth muscles of the gastrointestinal tract and abundant expression in several vascular smooth muscles. We propose that cKv1.5 represents a component of the delayed rectifier current in both vascular and visceral smooth muscles.

Amino Acid Sequence↗

PubMed1994

Sources of calcium utilized in cholinergic responses in canine colonic smooth muscle.

Ratiometric fura 2 fluorescence techniques were used to investigate the sources of Ca2+ that lead to an increase in cytosolic Ca2+ concentration ([Ca2+]i) and the generation of force during cholinergic stimulation of canine colonic circular smooth muscle tissues. Acetylcholine (ACh; 1 microM) caused a biphasic increase in [Ca2+]i and force. The initial upstroke phase was characterized by an increase in [Ca2+]i and a pronounced increase in force. The sustained phase was characterized by concurrent oscillations in [Ca2+]i and force (2-3/min) that persisted as long as ACh was present. The increase in [Ca2+]i in response to ACh was reduced to approximately 30% in the presence of nicardipine (1 microM), suggesting that L-type Ca2+ channels contribute to the rise in [Ca2+]i but that other sources also contribute. Preincubation in caffeine (10 mM) and ryanodine (10 microM) reduced the upstroke phase of the increase in [Ca2+]i and contractile responses to ACh, indicating that release of Ca2+ from intracellular stores contributes only to the initial cholinergic response. Responses to ACh persisted when nicardipine (1 microM) was included after emptying of caffeine-ryanodine-sensitive stores, suggesting the presence of additional sources of Ca2+. Data suggest that cholinergic regulation of [Ca2+]i in colonic smooth muscle occurs by a number of parallel pathways. Influx through voltage-dependent Ca2+ channels, release of Ca2+ from intracellular stores, and possibly Ca2+ entry through additional conductances activated by ACh all contribute to the regulation of [Ca2+]i.

Acetylcholine↗

PubMed1994

Oblique smooth muscle bundles between the circular and longitudinal muscle layers in the canine proximal colon.

The region between the longitudinal and circular muscle layers of the canine proximal colon was investigated by light and electron microscopy. Unique smooth muscle bundles, referred to as "oblique" muscles, were arranged in branching/anastomosing bundles, forming a network intermeshed with the nerve plexus and vasculature. These smooth muscles were concentrated along the ganglia and primary connectives of the myenteric plexus, thus showing a periodic distribution roughly corresponding to that of the ganglia. The oblique smooth muscle bundles bridged the distance between the circular and longitudinal muscle layers and blended into the inner aspect of the longitudinal muscle layer and the outer aspect of the circular muscle layer. A few oblique muscle cells occurred within the sheath of the ganglia and primary strands, i.e., perineurium. Oblique muscles may serve to electrically couple the circular and longitudinal muscle layers, thus explaining the synchronous electrical activities of these muscles. It is possible that the pacemaker activity in the myenteric region is generated by these muscle cells. Oblique muscles may also serve to protect nerve elements and the vasculature from the mechanical stress of peristalsis.

Animals↗

PubMed1994

Mechanism of cyclic AMP-induced hyperpolarization in canine colon.

The mechanism of forskolin (FSK)-induced hyperpolarization was investigated in strips of canine colonic circular muscle. FSK responses were compared to those of the K+ channel opener lemakalim (LEM). Both FSK (10 microM) and LEM (10 microM) hyperpolarized cells near the myenteric border by 10 to 20 mV. Responses to both agents were abolished by 35 mM external K+, indicating a probable mediation by K+ channels. FSK increased the open probability of Ca(++)-activated K+ channels in isolated colonic myocytes. However, in muscle strips charybdotoxin (100 nM) and tetraethylammonium (10 mM) failed to reduce FSK- and LEM-induced hyperpolarizations whereas tetrapentylammonium (50 microM) and 4-aminopyridine (10 mM) blocked both responses. Phencyclidine (100 microM), Ba++ (1 mM) and the antagonist of ATP-sensitive K+ currents glybenclamide (10 microM) blocked LEM- but not FSK-induced hyperpolarizations. Delayed rectifier current in isolated myocytes was activated near -20 mV and was blocked by (order of potency): nifedipine > tetrapentylammonium > phencyclidine > 4-aminopyridine > tetraethylammonium. Charybdotoxin (100 nM), Ba++ (1 mM) and glybenclamide (10 microM) were without effect. Ca(++)-activated K+ current was activated near +30 mV and was blocked by: charybdotoxin > tetraethylammonium > tetrapentylammonium >> phencyclidine = 4-amino-pyridine. These data suggest that LEM induces membrane hyperpolarization by activation of a K+ current with a pharmacology similar to ATP-sensitive K+ current whereas cyclic AMP-induced hyperpolarization appears to involve activation of a current other than delayed rectifier current, Ca(++)-activated K+ current or ATP-sensitive K+ current.

Animals↗

PubMed1993

Cloning and expression of a Kv1.2 class delayed rectifier K+ channel from canine colonic smooth muscle.

A cDNA (CSMK1) encoding a delayed rectifier K+ channel of the Kv1.2 class was cloned from canine colonic circular smooth muscle and expressed in Xenopus oocytes. These channels appear to be uniquely expressed in gastrointestinal muscles and may participate in the electrical slow wave activity. Functional expression of CSMK1 in Xenopus oocytes demonstrated a K+ current that activated in a voltage-dependent manner upon depolarization. This current was highly sensitive to 4-aminopyridine (IC50, 74 microM). A low-conductance K+ channel was identified in inside-out patches from oocytes injected with CSMK1. This channel displayed a linear current-voltage relation with a slope conductance of 14 pS. The channels were blocked in a concentration-dependent manner by 4-aminopyridine. Northern blot analysis demonstrated that CSMK1 is expressed in a wide variety of gastrointestinal smooth muscles. Portal vein, renal artery, and uterus do not express CSMK1, suggesting that, among smooth muscles, expression of this K+ channel may be restricted to gastrointestinal smooth muscles. CSMK1 is 91% homologous to RAK, a delayed rectifier K+ channel cloned from rat heart, but displays unique pharmacological properties and tissue distribution.

4-Aminopyridine↗

PubMed1993

Amplification of nitric oxide signaling by interstitial cells isolated from canine colon.

The effects of nitric oxide (NO) on intracellular Ca2+ concentration ([Ca2+]i) were studied in enzymatically dispersed interstitial cells (ICs) and smooth muscle cells (SMCs) isolated from canine colon. [Ca2+]i was monitored by using fluo-3 and video fluorescence imaging techniques. Exogenous NO caused an increase in [Ca2+]i in ICs and a decrease in [Ca2+]i in SMCs. Effects of NO on ICs were not blocked by removal of extracellular Ca2+ but were blocked by ryanodine, suggesting that NO caused release of Ca2+ from intracellular stores. When [Ca2+]i was elevated in an IC by micropressure ejection of Bay K 8644, [Ca2+]i decreased in nearby SMCs, suggesting release of a diffusible substance. The diffusible substance may be NO or an NO-related substance based on blockade of transmission by NG-nitro-L-arginine methyl ester, NG-monomethyl-L-arginine, or oxyhemoglobin. The elevation of [Ca2+]i in ICs by NO, which, in turn, might cause further release of NO and elevation of [Ca2+]i, suggests a positive feedback and amplification mechanism in these cells. Elevation of [Ca2+]i in SMCs had no effect on adjacent SMCs. Our data suggest that ICs may play a central role in amplification of NO signaling and propagation of inhibitory wave fronts.

Animals↗

PubMed1993

Identification of interstitial cells in canine proximal colon using NADH diaphorase histochemistry.

In gastrointestinal muscles special cells, referred to an interstitial cells, may be involved in pacemaking and transduction of inputs from the enteric nervous system. We have used a modification of the NADH diaphorase method to characterize the distribution of interstitial cells in the muscularis externa of the canine colon. The staining product of the NADH diaphorase reaction is useful because it allows light and electron microscopic studies to be performed with the same marker. Therefore rigorous identification of the cells observed at the light microscopic level could be made by electron microscopy. We were able to label at least three classes of interstitial cells: (1) at the submucosal surface of the circular muscle layer; (2) within the thickness of the circular and longitudinal muscle layers; and (3) in the region of the myenteric plexus. This technique also labeled cell bodies and initial segments of processes of Dogiel type II neurones in enteric ganglia. Nerve fibres within the muscle layers did not exhibit NADH diaphorase activity. This study has identified the interstitial cells within the circular and longitudinal muscle layers and shows the arrangement of these cells in a three-dimensional network.

Animals↗

PubMed1993

Enteric inhibitory neural regulation of human colonic circular muscle: role of nitric oxide.

BACKGROUND: Nitric oxide and an apamin-sensitive transmitter may both contribute to neural inhibition in the human colon. The present study investigated the role of NO in regulating spontaneous rhythmic contractions and examined NO-dependent and independent components of neurally evoked hyperpolarization in the human colon. METHODS: Mechanical and electrical activity were recorded from isolated circular muscle strips. RESULTS: Rhythmic contractions were inhibited by nerve stimulation. This response was reduced by apamin, oxyhemoglobin, and L-NG-nitro arginine methyl ester (L-NAME). Electrical recording revealed two components of neurally evoked hyperpolarization: a fast hyperpolarization resulting from a single stimulus and a sustained hyperpolarization that developed with repetitive stimulation. Fast hyperpolarization was not affected by L-NAME or oxyhemoglobin but was significantly reduced by apamin. The sustained hyperpolarization was reduced by L-NAME or apamin. Exogenous NO and the P2y receptor agonist 2-methylthio adenosine 5'-triphosphate (2-MATP) inhibited spontaneous contractions and produced hyperpolarization. Apamin reduced the effects of 2-MATP but not those of NO. CONCLUSIONS: The results support the concept that the inhibitory neurotransmission in the human colon involves two transmitters. A single stimulus results in an apamin-sensitive response. With multiple stimuli, a NO-dependent response develops and sums with the apamin-sensitive mechanism, producing sustained hyperpolarization and inhibition of contractions.

Action Potentials↗

PubMed1993

Immunohistochemical localization of 3',5'-cyclic guanosine monophosphate in the canine proximal colon: responses to nitric oxide and electrical stimulation of enteric inhibitory neurons.

There is growing evidence that nitric oxide serves as a neurotransmitter released from enteric inhibitory nerves in the gastrointestinal tract. The distribution of nitric oxide synthase suggests that nitric oxide may also be a neurotransmitter within enteric ganglia. Since many actions of nitric oxide are mediated by stimulation of soluble guanylate cyclase and a subsequent increase in 3',5'-cyclic guanosine monophosphate (cGMP) concentration, targets for nitric oxide in the canine proximal colon were investigated by immunohistochemical localization of cGMP. In the presence of phosphodiesterase inhibitors (M&B 22948, 100 microM and 3-isobutyl-1-methyl-xanthine, 1 mM), exogenous nitric oxide and electrical field stimulation caused an accumulation of cGMP-like immunoreactivity in several cell-types including colonic smooth muscle cells. cGMP-like immunoreactivity was also observed in a subpopulation of neurons in both myenteric and submucosal ganglia. Sequential labeling with the NADPH diaphorase technique showed that 94% of neurons that responded to exogenous nitric oxide with an increase in cGMP-like immunoreactivity were NADPH diaphorase negative. None of the myenteric neurons that responded to electrical field stimulation with an increase in cGMP-like immunoreactivity were NADPH diaphorase positive, and only one submucosal neuron with cGMP-like immunoreactivity was also NADPH diaphorase positive. The electrical field-stimulated increase in cGMP-like immunoreactivity was blocked by nitroarginine (100 microM). An increase in cGMP-like immunoreactivity also occurred in interstitial cells located at the submucosal surface of the circular muscle layer. These cells are interposed between nerve varicosities and smooth muscle cells and may partially mediate neuromuscular transmission. Sodium nitroprusside and nitric oxide also caused an accumulation of cGMP-like immunoreactivity in smooth muscle cells of intramural arterioles and venules. The results of this study further support the role of nitric oxide as a neurotransmitter in colonic muscles, and provide support for the hypothesis that interstitial cells are functionally innervated by enteric inhibitory neurons. The data also suggest that nitric oxide may serve as a neurotransmitter in enteric ganglia.

1-Methyl-3-isobutylxanthine↗

PubMed1993

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 delta-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 delta-specific antagonist. 5. MetEnk and DADLE blocked a portion of the i.j.p. that was sensitive to arginine analogues; after treatment with N omega-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 delta 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.

Animals↗

PubMed1993

Inhibition of nitric oxide synthesis reveals non-cholinergic excitatory neurotransmission in the canine proximal colon.

1. Neuromuscular transmission in the circular muscle of the canine proximal colon was examined, in the presence and absence of nitric oxide synthase inhibitors, by use of mechanical and intracellular microelectrode recording techniques. 2. Electrical field stimulation (EFS; 0.1-20 HZ) produced frequency-dependent contractions of circular muscle strips which reached a maximum at 15 Hz. These responses were enhanced by NG-monomethyl-L-arginine (L-NMMA; 300 microM) and reduced by atropine (1 microM). The effects of L-NMMA were reversed by L-arginine (3 mM). All responses to EFS were abolished by tetrodotoxin (1 microM). 3. In the presence of atropine, phentolamine and propranolol (all at 1 microM; 'non-adrenergic, non-cholingergic (NANC) conditions'), EFS evoked frequency-dependent inhibition of phasic contractions which reached a maximum at 5 Hz. At higher frequencies of EFS, inhibition diminished, and these responses were followed by post-stimulus excitation. 4. Under NANC conditions and in the presence of L-NG-nitroarginine methyl ester (L-NAME; 200 microM), EFS evoked contractions at frequencies of 5 Hz or greater. These contractions were reduced by co-incubation with L-arginine (2 mM) and abolished by tetrodotoxin (1 microM). 5. In the presence of atropine (1 microM), EFS (5-20 Hz) caused frequency-dependent inhibition of electrical slow waves. In the presence of L-NAME (100 microM) and atropine, the inhibitory response to EFS was abolished and an increase in slow wave duration was seen at stimulation frequencies greater than 5 Hz. The effects of EFS on slow wave duration were abolished by tetrodotoxin (1 microM). 6. Atropine-resistant contractions to EFS were enhanced by indomethacin (10 microM) and reduced or abolished by the non-selective NK1/NK2 tachykinin receptor antagonist D-Pro2, D-Trp7,9 SP, and by the selective NK2 receptor antagonist MEN 10,376 (10 microM).7. Exogenous tachykinins mimicked non-cholinergic excitatory electrical and mechanical responses. The rank order of potency for contraction was neurokinin A>neurokinin B>substance P, suggesting a predominance of the NK2 sub-type of tachykinin receptors on colonic smooth muscle cells. Low concentrations of neurokinin A also increased the amplitude and duration of electrical slow waves.8. These results suggest that: (i) in previous studies, non-cholinergic excitatory responses were masked by the simultaneous release of NO; (ii) non-cholinergic excitatory responses occur throughout the period of stimulation and are not manifest only as 'rebound' excitation; (iii) one or more tachykinins, possibly,acting via NK2 receptors, may mediate non-cholinergic excitatory responses.

Amino Acid Oxidoreductases↗

PubMed1993

Ca2+ regulation of the contractile apparatus in canine gastric smooth muscle.

1. The relationships between cytosolic Ca2+ ([Ca2+]cyt; expressed as a fluorescence ratio at 400 nm and 500 nm using Indo-1) and contractile force was examined in strips of circular smooth muscles of canine gastric antrum. Rhythmic increases in [Ca2+]cyt were observed and contractions were biphasic. 2. In most muscles (70%), the amplitude of the second phase of the Ca2+ transient was less than or equal to the first phase of the Ca2+ transient, but the second phase of the contraction was much smaller than the first phase, suggesting a decrease in Ca2+ sensitivity during the second contractile phase. In 30% of muscles, the amplitude of the second phase of the Ca2+ transient was 2- to 3-fold greater than the first phase. In these muscles, the second phase of contraction was 10-fold greater than the first phase of contraction. Thus, a non-linear relationship between [Ca2+]cyt and force greatly amplifies force development when [Ca2+]cyt exceeds a threshold level. 3. Acetylcholine (ACh, 0.3-1 microM) increased the amplitudes of Ca2+ transients and basal [Ca2+]cyt between phasic contractions. The increase in basal [Ca2+]cyt did not cause tone to develop. ACh increased the amplitude of Ca2+ transients 2- to 3-fold and this was associated with a 15 to 20-fold increase in the force of phasic contractions. Pentagastrin (0.5 nM) and cholecystokinin octapeptide (CCK, 40 nM) had similar effects on Ca2+ transients and phasic contractions. 4. Bay K 8644 (0.1 microM) and TEA (5 mM) also increased the amplitudes of Ca2+ transients by 2- to 3-fold and phasic contractions by 15- to 30-fold. There was no significant difference observed between the [Ca2+]cyt-force relationships in the presence of agonists (i.e. ACh, pentagastrin and CCK) or when [Ca2+]cyt was increased by Bay K 8644 or TEA. These data suggest that agonist-dependent increases in Ca2+ sensitivity may not significantly regulate the [Ca2+]cyt-force relationship in antral muscles. 5. D600 (5 microM), added during stimulation with ACh (0.3 M), decreased [Ca2+]cyt and force without affecting the [Ca2+]cyt-force relationship. 6. Mechanisms exist for agonist-mediated enhancement of the Ca(2+)-force relationship. In alpha-toxin-permeabilized antrum, ACh (10 microM) with GTP (100 microM) or GTP gamma S (100 microM) increased the Ca(2+)-induced contraction at clamped levels of Ca2+. Phorbol 12,13-dibutyrate (PDBu, 10 microM) also increased the contractile force at a given level of Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1993

Comparison of ionic currents from interstitial cells and smooth muscle cells of canine colon.

1. Voltage-dependent ionic currents of isolated interstitial cells were characterized using the whole-cell voltage clamp technique, and compared with currents recorded from circular muscle cells. Both cell types were isolated from the submucosal pacemaking region in the canine distal colon. 2. Upon depolarization, interstitial cells and smooth muscle cells generated transient inward, followed by slowly inactivating outward, currents. 3. After blocking inward current and much of the Ca(2+)-dependent outward current, interstitial cells displayed voltage-dependent outward current that rapidly activated, reached a peak, and then inactivated. This current was resistant to 4-aminopyridine(4-AP; 1 mM). Smooth muscle cells expressed a similar current but it was reduced by about 40% at a test potential of +20 mV by 4-AP (1 mM). 4. The inactivation characteristics of the voltage-dependent outward currents of interstitial cells and smooth muscle cells were compared. The outward current of interstitial cells inactivated at more negative potentials; half-inactivation occurred at -53 mV, whereas half-inactivation occurred at -20 mV in smooth muscle cells. 5. Inward currents were not strikingly different in the two cell types when dialysing pipettes were used. When the perforated patch technique (using Amphotericin-B) was used, a negatively activating inward current was observed in interstitial cells that had a resolution threshold of -70 to -60 mV. This current peaked at -10 mV. Inward currents in smooth muscle cells were resolved at test potentials positive to -50 mV and peaked at 0 to +10 mV. 6. When interstitial cells were held at -40 mV, inward current could not be resolved with test depolarization negative to -30 mV. From this holding potential, peak amplitude was reduced by 85% with test depolarizations to -10 mV. Holding smooth muscle cells at -40 mV also reduced inward current, but the peak current in these cells was reduced by only 39% at 0 mV. 7. Ni2+ partially inhibited peak inward current in interstitial cells and abolished a 'hump' in the I-V curve that occurred at negative potentials. In dialysed cells where this 'hump' was not apparent, addition of nifedipine unmasked a 'hump'. The presence of both nifedipine and Ni2+ abolished inward current. 8. A portion of the inward current in smooth muscle cells was sustained and persisted for the duration of test pulses. Very little sustained inward current was observed in interstitial cells. 9. The time course of inactivation of inward current in interstitial cells was fitted with two exponentials.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Aminopyridine↗

PubMed1993

Inhibition of slow-wave repolarization and Ca(2+)-activated K+ channels by quaternary ammonium ions.

We studied the effects of the K+ channel blocker tetrapentylammonium (TPeA) on the electrical activity of intact circular smooth muscle from canine colon. TPeA (10 and 20 microM) increased slow-wave duration and "locked" the membrane potential around -30 mV plateau potential after several minutes of application, suggesting that K+ channels are essential for termination of colonic slow waves. Repolarization and normal slow-wave activity resumed after 20-30 min of washout. The patch-clamp technique was used to study the block of large-conductance Ca(2+)-activated K+ channels (BK channels) by TPeA and tetraethylammonium (TEA) in excised and cell-attached patches from isolated colonic smooth muscle cells. Channel block was characterized by a voltage-dependent dissociation constant [Kd(V)] for the binding of TEA and TPeA to a blocking site located a fraction of the distance across the membrane field (delta). The extracellular TEA binding site had a Kd(0) of 0.33 mM and a delta of 0.23. The extracellular TPeA binding site had a Kd(0) of 2.2 mM but showed significantly less voltage dependence (delta = 0.02). The intracellular binding site for TEA was of low affinity [Kd(0) = 76 mM]. Intracellular TPeA was the most potent blocker of BK channel current [Kd(0) = 11.7 microM]. The voltage dependence of block by intracellular TPeA (delta = -0.21) was not significantly different from that of intracellular TEA (delta = -0.3). Internal TPeA (10 microM) also blocked a 70-pS K+ channel and a 23-pS K+ channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia↗

PubMed1993

Dihydropyridine-sensitive calcium channels expressed in canine colonic smooth muscle cells.

Experiments were performed to identify and characterize the types of calcium channels that regulate inward calcium current in canine colonic smooth muscle. Freshly dispersed smooth muscle cells from the circular layer of the canine proximal colon were used. Single-channel currents were measured with 80 mM Ba2+ as the charge carrier. Small-conductance (10 +/- 2 pS, EBa = 46 +/- 11 mV, n = 9) and large-conductance (21 +/- 1 pS, EBa = 52 +/- 3 mV, n = 19) single-channel currents were observed during depolarizing voltage steps positive to -30 mV. Both types of single-channel currents were inhibited by the addition of 10(-6) M nifedipine to the bath solution. The smaller current was infrequently observed and therefore was not further characterized. Open probability (P(o)) of the larger current amplitude was strongly dependent on voltage. Activation curves were well described by a Boltzmann function with half activation occurring at 4 mV, and a 5-mV increase in membrane potential resulted in an e-fold increase in P(o). BAY K 8644 (1 microM) shifted the activation curve to the left while nifedipine (1 microM) resulted in a right shift. Molecular analysis showed that only the C class of Ca2+ channel alpha 1-subunit is expressed in this tissue. Furthermore, only a single splice variant (rbc-II) was observed. The results suggest that a single class of dihydropyridine-sensitive calcium channels regulates inward calcium current in canine colonic smooth muscle cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗