Biomedical subjects

S M Sims

Publications and source records attributed to S M Sims.

At least 37 records · Page 2Linked to original sources

PubMed1998

Wortmannin inhibits spreading and chemotaxis of rat osteoclasts in vitro.

Wortmannin (WT) and 17beta-hydroxywortmannin (HWT), which are inhibitors of phosphatidylinositol-3(OH)-kinase (PI3K), have been shown previously to inhibit bone resorption in vitro and in vivo, possibly by interfering with formation of the osteoclast ruffled border. Since migration of osteoclasts also plays an important role in the process of bone resorption, we investigated the effects of these inhibitors on osteoclast morphology and motility. Both HWT and WT caused a sustained decrease in the planar area of osteoclasts in vitro (half maximal effect at 25 and 165 nM, respectively), with the effect of HWT on cell area more readily reversible than WT. These agents also caused accumulation of intracellular vesicles. Time-lapse video microscopy was used to record the migration of osteoclasts in response to macrophage colony-stimulating factor (M-CSF) or vehicle, flowing passively from a micropipette positioned 200-400 microm from the cell. M-CSF caused directed migration of osteoclasts, indicating chemotaxis (over 3 h osteoclasts migrated 96 +/- 14 microm in response to M-CSF vs. 11 +/- 2 microm in control experiments). Both WT (100 or 500 nM) and LY294002 (100 microM), a specific PI3K inhibitor structurally unrelated to WT, significantly inhibited osteoclast chemotaxis in response to M-CSF. Taken together, these effects of WT, HWT, and LY294002 are consistent with an important role for PI3K in regulating cytoskeletal function in osteoclasts. The inhibitory effects of WT and HWT on bone resorption may be due, in part, to impairment of osteoclast motility.

Androstadienes↗

PubMed1998

Hydrogen peroxide-induced stimulation of L-type calcium current in guinea pig ventricular myocytes and its inhibition by adenosine A1 receptor activation.

Hydrogen peroxide (H2O2) produces complex cardiac effects that may involve altered calcium homeostasis. The cardiotoxic effects of H2O2 can be attenuated by adenosine A1 receptor agonists. The present study examined the effect of H2O2 on L-type Ca++ current (ICa,L) in guinea pig ventricular myocytes under two different recording conditions and the influence of adenosine receptor agonists. H2O2 (100 microM), did not have any significant effect on ICa,L, under conventional whole cell patch configuration. However, when recorded under nystatin perforated patch configuration, H2O2 caused a gradual and significant increase (84 +/- 14%) in ICa,L compared to control values. N6-cyclopentyladenosine (CPA), an adenosine A1 receptor agonist, significantly attenuated the effect of H2O2. The inhibitory effect of N6-cyclopentyladenosine was antagonized by 8cyclopentyl-1, 3-dipropylxanthine, an adenosine A1 receptor antagonist. The A2A and A3 receptor agonists, 2-p-(2-Carboxyethyl)phenethylamino-5'- N - ethylcarboxamidoadenosine (CGS-21680) and 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-be ta-D-ribofuranuronamide, respectively, did not modulate the enhancement of ICa,L by H2O2. Moreover the effects of N6-cyclopentyladenosine were mimicked by the protein kinase C inhibitor bisindolylmaleimide. Thus, our results demonstrate a potent stimulatory effect of H2O2 on ICa,L in guinea pig ventricular myocytes. We further demonstrate that adenosine A1 receptor activation attenuates this effect. Our results suggest a potential basis for altered calcium homeostasis in response to H2O2 as well as the salutary effects of A1 receptor activation against H2O2-induced cardiotoxicity.

Adenosine↗

PubMed1997

Extracellular nucleotides activate non-selective cation and Ca(2+)-dependent K+ channels in rat osteoclasts.

1. Extracellular ATP elevates cytosolic free Ca2+ concentration ([Ca2+]i) in osteoclasts, but its effects on ion channels have not been reported previously. Membrane currents and [Ca2+]i were recorded in isolated rat osteoclasts using patch clamp and fluorescence techniques. 2. At negative membrane potentials, ATP (1-100 microM) activated an inward current that peaked rapidly and then declined. A later current was outward at potentials positive to the equilibrium potential for K+ (EK) and showed oscillations. 3. The initial inward current, studied in isolation using Cs+ in the electrode solution, showed rapid activation, inward rectification and reversal at +3 +/- 4 mV. Reduction of [Na+]o to 10 mM shifted the reversal potential to -21 +/- 3 mV, indicating that ATP activates a non-selective cation current, consistent with involvement of P2X receptors. 4. The later current activated by ATP, studied with K+ in the electrode solution, exhibited a linear I-V relationship, and reversed at -71 +/- 4 mV. The reversal potential shifted 51 mV per 10-fold change of [K+]o, indicating that ATP activates a K+ current (IK). 5. In fura-2-loaded cells, ATP caused elevation of [Ca2+]i that persisted in Ca(2+)-free solution, indicating that ATP induced release of Ca2+ from intracellular stores, consistent with involvement of P2Y receptors. Simultaneous patch clamp and fluorescence recordings revealed that IK was associated with the elevation of [Ca2+]i. Using a Ca2+ ionophore (4Br-A23187) to elevate [Ca2+]i, IK activated when [Ca2+]i exceeded approximately 400 nM, with half-maximal activation at 580 +/- 50 nM. 6. In cell-attached patches, ATP activated a channel with a conductance of 48 +/- 6 pS, that reversed director, near EK. Channel open probability increased with elevation of [Ca2+]i, indicating the Ca2+ dependence of this channel. 7. These results demonstrate that rat osteoclasts express two types of purinoceptors. P2X receptors give rise to non-selective cation current. P2Y receptors mediate Ca2+ release from stores, causing activation of a Ca(2+)-dependent K+ channel.

Adenosine Triphosphate↗

PubMed1997

Differential effects of endothelin-1 on basal and isoprenaline-enhanced Ca2+ current in guinea-pig ventricular myocytes.

1. We examined the effect of endothelin-1 (ET-1) on basal and isoprenaline-enhanced L-type Ca2+ current (ICa,L) in guinea-pig ventricular myocytes under nystatin-perforated patch configuration. 2. ET-1 at concentrations of 1, 5 and 10 nM had little effect on basal ICa,L. However, ICa,L enhanced by isoprenaline (500 nM) was significantly attenuated by 5 nM ET-1 by more than 50%. This effect was reversed upon washout. ICa,L enhanced by forskolin was also decreased by ET-1. 3. The inhibitory effect of ET-1 against isoprenaline was completely blocked by the ETA receptor antagonist BQ-123 (1 microM). In myocytes incubated with pertussis toxin (PTX, 2 micrograms ml-1) for 5 h, ET-1 did not inhibit isoprenaline-enhanced ICa,L. 4. Although ET-1 has been shown to activate specific protein kinase C (PKC) isoforms, a significant inhibitory effect of ET-1 was maintained in the presence of the PKC inhibitor bisindolylmaleimide (20 nM). The nitric oxide (NO) donor SIN-1 (10 microM) attenuated but failed to prevent the ET-1 effect. 5. In summary, our results demonstrate that ET-1 is devoid of any significant effects on basal ICa,L. However, it exerts a potent inhibitory effect against isoprenaline-enhanced ICa,L. This effect is mediated through ETA receptors coupled to PTX-sensitive G-proteins and occurs in the presence of PKC inhibition and NO generation.

Animals↗

PubMed1997

Regulation of intracellular calcium in human esophageal smooth muscles.

We have investigated sources of Ca2+ contributing to excitation of human esophageal smooth muscle, using fura 2 to study cytosolic free Ca2+ concentration ([Ca2+]i) in dispersed cells and contraction of intact muscles. Acetylcholine (ACh) caused an initial peak rise of [Ca2+]i followed by a plateau accompanied by reversible contraction. Removal of extracellular Ca2+ or addition of dihydropyridine Ca2+ channel blockers reduced the plateau phase but did not prevent contraction. Caffeine also caused elevation of [Ca2+]i and blocked responses to ACh. Undershoots of [Ca2+]i were apparent after ACh or caffeine. Blockade of the sarcoplasmic reticular Ca(2+)-ATPase by cyclopiazonic acid (CPA) reduced the ACh-evoked increase of [Ca2+]i and abolished the undershoot, indicating involvement of Ca2+ stores. When contraction was studied in intact muscles, removal of Ca2+ or addition of nifedipine reduced, but did not abolish, carbachol (CCh)-induced contraction. Elevation of extracellular K+ caused contraction that was inhibited by nifedipine, although CCh still elicited contraction. CPA caused contraction and suppressed the CCh-induced contraction, whereas ryanodine reduced CCh-induced contraction. Our studies provide evidence that muscarinic excitation of human esophagus involves both release of Ca2+ from intracellular stores and influx of Ca2+.

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

PubMed1996

Cholinergic inhibition of Ca2+ current in guinea-pig gastric and tracheal smooth muscle cells.

1. Cholinergic regulation of L-type Ca2+ channels was investigated in freshly dissociated guinea-pig gastric and tracheal smooth muscle cells. Acetylcholine (ACh, 50 microM) decreased Ca2+ channel current (ICa) by 37 +/- 3% (mean +/- S.E.M., 46 cells). 2. ACh reduced ICa at all voltages, with no shift in the current-voltage relationship. Effects of ACh were rapid (within 5 s) and repeatable, with multiple applications reproducibly inhibiting ICa in the continued presence of extracellular Ca2+ and in the presence of protein kinase C inhibitors. 3. The involvement of Ca2+ stores in this inhibition was investigated using Ca(2+)-free solution or cyclopiazonic acid (CPA) to deplete the stores. ACh initially inhibited ICa in the Ca(2+)-free solution (Na+ as charge carrier, 53 +/- 4% decrease, 18 cells) with subsequent responses significantly attenuated (n = 9). CPA (1 microM) reduced, then abolished, the effects of ACh on ICa (n = 5). 4. When studied in cell-attached patches (Ba2+ as charge carrier), ACh reduced Ca2+ channel open probability in twenty-two of thirty-six cells, consistent with the involvement of a diffusible cytosolic messenger. 5. ACh also inhibited ICa in tracheal muscle cells (reduction of 38 +/- 6% in 1 mM Ca2+, 4 cells; 77 +/- 3% in Ca(2+)-free solution, 7 cells). Furthermore, in cells where ACh elicited oscillating Ca(2+)-activated Cl- current, oscillatory inhibition of ICa was also observed (3 cells). 6. In summary, ACh causes rapid and reversible inhibition of ICa in gastric and tracheal muscles. Ca2+ stores were required to initiate this effect, with the rapid onset and oscillatory inhibition consistent with Ca2+ inhibition of the channel. Suppression of ICa would reduce Ca2+ entry during cholinergic excitation.

Acetylcholine↗

PubMed1996

Influence of organic acid excretion on cuticle pH and drug absorption by Haemonchus contortus.

To determine if a cuticle microenvironment pH is maintained by adult Haemonchus contortus, organic acid excretion kinetics and absorption kinetics of selected model weak acids and a weak base were measured in incubation media that varied in buffer capacity (0.25-20 mM HEPES or 5 mM glycine) and initial pH (7.5 or 3.5). To evaluate the importance of the cuticle as a pathway for organic acid excretion and drug absorption the pharynx was paralyzed with 1 nM ivermectin. H. contortus changed the media pH from initial values of 7.5 or 3.25 to an asymptotic value of approximately 5.6. The rate of pH change depended on the buffer capacity, but was not affected by chemical ligation with ivermectin. The intrinsic rate of excretion of organic acids (0.045 +/- 0.016 micromol/cm2 x h) was constant during the first 8-12 h of incubation and was independent of initial pH, buffer capacity or ivermectin ligation. The rates of absorption of the model weak acids, benzoic acid and p-nitrophenol, and the model weak base, aniline, were not affected by initial pH, buffer capacity or ivermectin ligation. These results suggest that H. contortus excretes organic acid endproducts of carbohydrate metabolism across its cuticle, and that these acids maintain a microenvironment pH within the water-filled pores of the cuticle that controls the rate of adsorption of weakly acidic or basic drugs.

Absorption↗

PubMed1996

Osteoclast activation in inflammatory periodontal diseases.

OBJECTIVE: In this paper, we review the mechanisms thought to be involved in the activation of osteoclasts in periodontitis. SUMMARY: Osteoclasts are regulated by both microbial and host factors. Some factors act directly on cells of the osteoclast lineage, whereas others act indirectly through other cell types in the bone environment. The proinflammatory cytokines (interleukins 1 and 6, tumor necrosis factors) have been implicated in the stimulation of osteoclastic resorption. The roles of the immunoregulatory cytoknes (interleukins 2 and 4, interferon gamma) are less clear, but decreased levels of these factors may contribute to periodontitis. A number of lipid mediators may be involved in stimulation of bone resorption. These include bacterial lipopolysaccharide and host-derived platelet-activating factor and prostaglandins. More recently, reactive oxygen intermediates and extracellular nucleotides, both present at sites of inflammation, have been investigated as possible modulators of osteoclast activity. The potential use of antiresorptive therapies in periodontitis is reviewed. CONCLUSIONS: A wide range of host and bacterial factors contribute to the loss of alveolar bone in periodontitis. However, much remains to be understood about the complex mechanisms through which these factors regulate osteoclast activity. Further studies at the cellular and molecular level will lead to a better understanding of these processes and perhaps suggest new approaches for periodontal therapy.

Alveolar Bone Loss↗

PubMed1996

Intracellular calcium stores in isolated tracheal smooth muscle cells.

We investigated the effects of acetylcholine (ACh) and histamine on intracellular calcium concentration ([Ca2+]i) and contraction of freshly isolated guinea pig tracheal smooth muscle cells. Previous electrophysiological studies revealed that agonists elicit cation and Cl- currents, but a role for Ca2+ in mediating these effects remains unresolved. Here we characterize agonist-induced changes of [Ca2+]i, using fura 2, and examine the contribution of the sarcoplasmic reticulum (SR) to regulation of [Ca2+]i. We provide evidence that the rise of [Ca2+]i and the contraction elicited by ACh or histamine are largely due to release of Ca2+ from stores. Agonists elicited Ca2+ transients in Ca(2+)-free solution with 0.5 mM ethylene glycol-bis (beta-aminoethyl ether)-N, N, N',N'-tetraacetic acid (EGTA), whereas prolonged exposure to Ca(2+)-free solution diminished the rise of [Ca2+]i. In addition, blockade of SR Ca(2+)-adenosinetriphosphatase (ATPase) by cyclopiazonic acid (CPA) or thapsigargin caused elevation of [Ca2+]i and reduction of ACh-evoked increase of [Ca2+]i. In many cells, [Ca2+]i fell below baseline (undershoot) after ACh or caffeine. CPA abolished this undershoot and reduced the rate of recovery of [Ca2+]i to basal levels. Furthermore, oscillations of [Ca2+]i were elicited in the presence or absence of extracellular Ca2+, and these too were reversibly abolished by CPA. Our results provide evidence that Ca2+ stores play a significant role in agonist-mediated increase of [Ca2+]i in tracheal muscle and that the SR contributes to the restoration of basal Ca2+ levels.

Acetylcholine↗

PubMed1995

Ca(2+)-dependent Cl- current in canine tracheal smooth muscle cells.

Our goal was to investigate the role of Ca2+ entry in regulating Cl- current (ICl) in smooth muscle cells from canine trachealis. When studies were done using the perforated patch configuration, depolarization elicited a dihydropyridine-sensitive Ca2+ current (ICa), followed in many cells by a sustained current. This sustained current reversed direction close to the Cl- equilibrium potential, consistent with its representing ICl. The ICl was also apparent as slowly deactivating tail currents seen upon repolarization to negative potentials. The Cl- channel blocker niflumic acid abolished both the sustained and tail currents, without affecting ICa. Several observations indicated that the ICl was dependent on Ca2+ entry. ICl was increased in magnitude when Ca2+ influx was augmented [by prolonging the depolarization or using BAY K 8644 or acetylcholine (ACh)] and decreased in magnitude when Ca2+ influx was reduced (using nifedipine). Based on these findings, we conclude that depolarization causes Ca2+ entry, with resultant elevation of cytosolic free Ca2+ concentration leading to activation of ICl (ICl(Ca)). We investigated whether Ca(2+)-induced Ca2+ release from the sarcoplasmic reticulum was involved in activation of ICl(Ca), by depleting intracellular stores of Ca2+ using cyclopiazonic acid to block the sarcoplasmic Ca(2+)-adenosinetriphosphatase and repeated stimulation with ACh. In such Ca(2+)-depleted cells, depolarization-mediated Ca2+ entry continued to activate ICl(Ca), suggesting that Ca(2+)-induced Ca2+ release was not required for its activation. We conclude that Ca2+ entry can activate Cl- channels in tracheal smooth muscle. This represents a positive-feedback system, which would promote excitation and contraction of airway muscle.

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

PubMed1994

Outwardly rectifying chloride current in rabbit osteoclasts is activated by hyposmotic stimulation.

1. We characterized chloride currents in freshly isolated rabbit osteoclasts using whole-cell and single channel patch-clamp recording configurations. Depolarization activated an outwardly rectifying current in 40-50% of cells, distinct from the inwardly rectifying K+ current we have previously reported in osteoclasts. 2. The outwardly rectifying current persisted under conditions where all K+ currents were blocked. Furthermore, the outward current was reversibly inhibited by Cl- transport blockers 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS); 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS); 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS); and niflumic acid. The blocked current had a reversal potential close to the predicted chloride equilibrium potential and was dependent on the chloride concentration gradient. 3. In those osteoclasts in which outwardly rectifying current was not initially apparent, exposure to hyposmotic extracellular solution resulted in its reversible activation. The induced current was due to Cl-, based on its reversal close to the chloride equilibrium potential and sensitivity to blockade by Cl- channel inhibitors. The hyposmotically induced current could be activated in Ca(2+)-free solutions containing 0.2 mM EGTA. 4. When studied in the current-clamp configuration, hyposmotic stimulation caused depolarization from -76 +/- 5 to -5 +/- 6 mV (mean +/- S.D., n = 7). 5. Unitary Cl- currents were recorded in the cell-attached patch configuration at positive potentials. Single channels had a slope conductance of 19 +/- 3 pS (n = 5). Reduction of the external [Cl-] shifted the current-voltage relationship in the positive direction, supporting the conclusion that these were Cl- currents. Like the whole-cell currents, single channel Cl- currents were activated by exposure of cells to hyposmotic bathing solution. 6. We conclude that rabbit osteoclasts express an outwardly rectifying Cl- current that can be activated by osmotic stress. Cl- channels may play a role in cell volume regulation and may also provide conductive pathways for dissipating the potential difference that arises from electrogenic proton transport during bone resorption.

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

PubMed1994

Murine osteoclasts and spleen cell polykaryons are distinguished by mRNA phenotyping.

To probe osteoclast gene expression, we combined the techniques of cell microisolation and RT-PCR to develop a novel and sensitive method for the isolation and mRNA phenotyping of small numbers of authentic osteoclasts and spleen cell polykaryons. Using this method we report (1) direct evidence for the presence of calcitonin receptor mRNA in osteoclasts, (2) confirmation of the recent finding of osteopontin mRNA in osteoclasts, and (3) demonstration that the specific expression of mRNA for tartrate-resistant acid phosphatase, carbonic anhydrase II, calcitonin receptor, and osteopontin enable one to distinguish the osteoclast from the morphologically similar and developmentally related spleen cell polykaryon. We also show that mRNA associated with the osteoblast phenotype, such as alkaline phosphatase, osteocalcin, and type I collagen, are absent in osteoclasts. This is the first report in which such an approach has been used successfully to distinguish the mRNA expression pattern of an authentic osteoclast from a macrophage polykaryon, and as such it should provide an important new tool for evaluating the results of various cell culture model systems designed to examine the origin and ontogeny of osteoclasts. Our results also indicate that these procedures can be used as an alternative to in situ hybridization methods for the cell-specific localization of specific mRNA in a mixed cell preparation and for colocalization of multiple mRNA species to a single cell type.

Animals↗

PubMed1994

Platelet-activating factor induces pseudopod formation in calcitonin-treated rabbit osteoclasts.

We demonstrated previously that platelet-activating factor (PAF), a potent inflammatory mediator, acts on osteoclasts to elevate cytosolic [Ca2+] and stimulate resorption. However, it is not clear whether the effects of PAF on resorptive activity are direct or indirect. In the present study, we investigated the effects of PAF on osteoclast motility. Osteoclasts were isolated from the long bones of neonatal rabbits, and cell motility and morphology were monitored using time-lapse video microscopy. Calcitonin, a hormone known to induce retraction of pseudopods and inhibit resorptive activity, was used to render osteoclasts quiescent. Within 10 minutes of calcitonin treatment (100 ng/ml, final), pronounced retraction of pseudopods was observed in 68 of 112 cells tested. When PAF (200 nM, final) was added 10 minutes after calcitonin treatment, pseudopods were evident 1 h later in 15 of 37 calcitonin-responsive cells tested. In contrast, pseudopods were evident in only 4 of 31 calcitonin-responsive cells treated with control solutions (PAF-vehicle or S-PAF, the biologically inactive stereoisomer of PAF). Pseudopod formation was quantified by measuring the planar area of pseudopods with a computer-based video analysis system. When assessed 60 minutes following PAF treatment, the pseudopod area was significantly greater in PAF-treated cells than in control cells. In some calcitonin-treated osteoclasts, PAF induced pseudopod formation when applied focally using an extracellular micropipette, consistent with a direct action of PAF. We conclude that PAF directly induces pseudopod formation in calcitonin-inhibited osteoclasts, a morphologic response indicative of osteoclast activation.

Animals↗

PubMed1994

Theoretical perspectives on anthelmintic drug discovery: interplay of transport kinetics, physicochemical properties, and in vitro activity of anthelmintic drugs.

This multidisciplinary study demonstrates the utility of the biophysical model approach to assess biological activity of anthelmintics in light of drug-delivery principles. The relationships between drug absorption and efficacy for a set of structurally disparate anthelmintics were determined in cultures of Haemonchus contortus, a nematode that parasitizes the ruminant gastrointestinal tract. Uptake, parameterized by the permeability coefficient, Pe, was shown to occur by absorption across the cuticle. Rates of drug appearance in nematode carcasses paralleled rates of drug disappearance from the medium, and absorption reached an apparent equilibrium within a few hours. The parasite/medium partition coefficient, K, was derived from the ratio of drug concentration in the parasite vs the medium at equilibrium. Pe and K values for each anthelmintic were correlated with lipophilicity (as measured by the partition coefficient (PC) in n-octanol/water) and both parameters plateaued at log PC approximately 2.5, with maximum Pe approximately 8 x 10(-4) cm/min and log K < or = 2.0. Absorption kinetics were related to in vitro potency by monitoring motility of H. contortus. The time required to reduce motility by 50% (t* 50) and Pe were used to calculate Cn*, the drug concentration in the parasite at t* 50, as an indicator of intrinsic potency. The quantitative interplay of apparent biological activity expressed as t* 50, dose, and intrinsic potency highlights the important contribution of drug-uptake kinetics.

Absorption↗

PubMed1994

Effects of extracellular calcium and protons on osteoclast potassium currents.

During resorption of mineralized tissues, osteoclasts are exposed to marked changes in the concentration of extracellular Ca2+ and H+. We examined the effects of these cations on two types of K+ currents previously described in these cells. Whole-cell patch clamp recordings of membrane currents were made from osteoclasts freshly isolated from neonatal rats. In control saline (1 mM Ca2+, pH 7.4), the voltage-gated, outwardly rectifying K+ current activates at approximately -45 mV and the conductance is half-maximally activated at -29 mV (V0.5). Increasing [Ca2+]out rapidly and reversibly shifted the current-voltage (I-V) relation to more positive potentials. Current at -29 mV decreased to 28 and 9% of control current at 5 and 10 mM [Ca2+]out, respectively. This effect of elevating [Ca2+]out was due to a positive shift of the K+ channel voltage activation range. Zn2+ or Ni2+ (5 to 500 microM) also shifted the I-V relation to more positive potentials and had additional effects consistent with blockade of the K+ channel. Based on the extent to which these divalent cations affected the voltage activation range of the outwardly rectifying K+ current, the potency sequence was Zn2+ > Ni2+ > Ca2+. Lowering or raising extracellular pH also caused shifts of the voltage activation range to more positive or negative potentials, respectively. In contrast to their effects on the outwardly rectifying K+ current, changes in the concentration of extracellular H+ or Ca2+ did not shift the voltage activation range of the inwardly rectifying K+ current.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Spontaneous transient inward currents and rhythmicity in canine and guinea-pig tracheal smooth muscle cells.

Spontaneous transient inward currents (STICs) were recorded in canine and guinea-pig tracheal myocytes held at negative membrane potentials. STICs were Cl- selective since their reversal potential was dependent on the Cl- gradient and they were blocked by the Cl- channel blocker niflumic acid. STICs were insensitive to Cs+, charybdotoxin, and nifedipine. Ca(2+)-activated K+ currents often preceded STICs, suggesting that the STICs are Ca2+ dependent. In support of this suggestion, we found the Cl- currents were: (1) abolished by depleting intracellular Ca2+ stores using caffeine, acetylcholine, histamine, or substance P; (2) enhanced by increasing external concentrations of Ca2+; (3) evoked by voltage-dependent Ca2+ influx. The channels responsible for this Cl- current are of small unitary conductance (< 20 pS). Decay of the STICs was described by a single exponential with a time constant of 94 +/- 9 ms at -70 mV; the time constant increased considerably at more positive potentials. Using Ca(2+)-dependent Cl- currents and contractions as indices of internal levels of Ca2+, we found that isolated tracheal cells are capable of exhibiting rhythmic behaviour: bursts of currents and contractions with a periodicity of less than 0.1 Hz and which continued for more than 20 min. These rhythmic events were recorded at negative membrane potentials, suggesting that cyclical release of internally sequestered Ca2+ is responsible. We conclude that spontaneous release of Ca2+ from intracellular stores in tracheal muscle cells leads to transient currents in some cases accompanied by rhythmic contractions. Our studies provide evidence for a cellular mechanism that could underly myogenic oscillations of membrane potential in smooth muscle.

Animals↗

PubMed1994

Substance P activates Cl- and K+ conductances in guinea-pig tracheal smooth muscle cells.

Substance P (SP) causes bronchoconstriction, but its effects on airway smooth muscle ion conductances are unknown. We investigated the effects of SP on single smooth muscle cells dissociated from guinea-pig trachealis. Under voltage clamp at -60 mV, SP evoked reversible contractions and inward current (ISP). ISP had a latency of approximately 1 s, reached a peak of 1039 +/- 147 pA (n = 19) about 2 s after onset of application, and declined to baseline levels over the next 5-10 s. At more positive holding potentials (-25 and 0 mV), the inward current was decreased in magnitude and preceded by outward current. With 140 mM K+ in the electrode and Cl- equilibrium potential (ECl) of about 0 mV, ISP was outwardly rectifying and reversed at -11 +/- 2 mV. When K+ currents were blocked using Cs+, the current-voltage relationship for ISP was linear and reversed at 3 +/- 1 mV. The reversal potential was dependent on the Cl- gradient across the membrane. These results suggest that SP caused a transient activation of Cl- and K+ conductances. Following the initial transient inward current, SP caused a prolonged suppression of spontaneously active K+ currents. The findings that SP evoked contractions during voltage clamp at potentials at which voltage-dependent Ca2+ channels are not active, and that current oscillations were also evoked by SP, suggest that SP is acting through release of Ca2+ from internal stores. Furthermore, SP occluded the inward current evoked by acetylcholine, suggesting that the peptidergic and cholinergic signalling pathways converge.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1994

Biophysical model of the transcuticular excretion of organic acids, cuticle pH and buffer capacity in gastrointestinal nematodes.

A biophysical model was developed, using Ascaris suum as a model gastrointestinal nematode, to provide quantitative perspectives into the microenvironmental pH within the water-filled, porous, negatively charged cuticle matrix of gastrointestinal nematodes. The central features of the model include (a) the constant rate of excretion of organic acid metabolites across the cuticle, (b) the relationship between cuticle pH and pKa of the organic acids that determines the fraction of unionized and ionized species, and (c) the concentration gradient, mean concentration and buffer capacity within the cuticle that maintain the cuticle pH. The model may be used to predict the extent to which transcuticular absorption of weakly basic and acidic anthelmintics will be affected by transcuticular excretion of organic acid metabolites. Coupled with established models for drug absorption by nematodes and the host gastrointestinal tract, the cuticle pH model provides new insights to the design of drugs with physicochemical properties that favor absorption by nematodes.

Acids↗