Plasma membrane electron transport in frog blood vessels

In an attempt to see if frog blood vessels possess a plasma membrane electron transport system, the postcaval vein and aorta isolated from Rana tigrina were tested for their ability to reduce ferricyanide, methylene blue, and 2,6-dichloroindophenol. While the dyes remained unchanged, ferricyanide was reduced to ferrocyanide. This reduction was resistant to inhibition by cyanide and azide. Heptane extraction or formalin fixation of the tissues markedly reduced the capability to reduce ferricyanide. Denuded aortas retained only 30% of the activity of intact tissue. Our results indicate that the amphibian postcaval vein and aorta exhibit plasma membrane electron transport.     

Interstitial cells in the vasculature

Interstitial cells of Cajal are believed to play an important role in gastrointestinal tissues by generating and propagating electrical slow waves to gastrointestinal muscles and/or mediating signals from the enteric nervous system. Recently cells with similar morphological characteristics have been found in the wall of blood vessels such as rabbit portal vein and guinea pig mesenteric artery. These non-contractile cells are characterised by the presence of numerous processes and were easily detected in the wall of the rabbit portal vein by staining with methylene blue or by antibodies to the marker of Interstitial Cells of Cajal c-kit. These vascular cells have been termed "interstitial cells" by analogy with interstitial cells found in the gastrointestinal tract. Freshly dispersed interstitial cells from rabbit portal vein and guinea pig mesenteric artery displayed various Ca2+-release events from endo/sarcoplasmic reticulum including fast localised Ca2+ transients (Ca2+ sparks) and longer and slower Ca2+ events. Single interstitial cells from the rabbit portal vein, which is a spontaneously active vessel, also demonstrated rhythmical Ca2+ oscillations associated with membrane depolarisations, which suggests that in this vessel interstitial cells may act as pacemakers for smooth muscle cells. The function of interstitial cells from the mesenteric arteries is yet unknown. This article reviews some of the recent findings regarding interstitial cells from blood vessels obtained by our laboratory using electron microscopy, immunohistochemistry, tight-seal patch-clamp recording, and fluorescence confocal imaging techniques.     

新鲜分离小鼠胃ICC样细胞的鉴定及其电生理学特性

将免疫荧光及传统全细胞膜片钳技术应用于新鲜分离的小鼠胃Cajal 间质细胞样细胞上,探讨了小鼠胃Cajal 间质细胞样细胞形态和电生理学特性。经胶原酶消化得到的Cajal间质细胞样细胞胞体呈短梭形,且自胞体发出多个较短的毛刺状突起。免疫细胞化学结果表明,Cajal 间质细胞样细胞胞体和突起酪氨酸激酶受体c-kit表达呈阳性。在传统全细胞记录模式、膜电位钳制在-60 mV 的条件下,可以记录到自发、节律性内向电流,即起搏电流。钙调蛋白抑制剂W-7 (50µmol/L）明显增强了起搏电流幅度并引发明显的内向钳制电流。当电极内液中EGTA 的浓度由0.1 mmol/L增加到10 mmol/L时,也明显增强了起搏电流幅度并引发明显的内向钳制电流。实验结果提示,新鲜分离的小鼠胃Cajal 间质细胞样细胞可以产生自发、自律性内向电流,而这种电流对胞内低钙或钙调蛋白抑制剂敏感。这种具有自发性电活动的Cajal 间质细胞样细胞可能就是胃Cajal 间质细胞。     

Antagonistic actions of prostaglandins E2 and F2 alpha on the isolated lungs of the frog, Rana esculenta L

Isolated lungs of the frog, Rana esculenta L., when incubated in amphibian Ringer solution for 30 min, produced a prostaglandin E2-like substance (27.1 +/- 3.8 ng/g w.w.), as determined by bioassay on the isolated rat stomach strip. The release of PGE2-like substance from skin, heart and bowel is also reported. The activity of synthetic prostaglandins E2 (PGE2) and F2 alpha (PGF2 alpha) on the muscular contractility of frog isolated lungs was investigated: PGE2 and PGF2 alpha relaxed and contracted respectively in a dose-dependent manner this preparation, a result similar to that obtained in mammals.     

Identification of interstitial cells of Cajal in the rabbit portal vein

Two layers of interstitial cells (ICs) of Cajal were detected by c-kit and methylene blue staining in the media of the rabbit portal vein in subendothelial intramuscular and deeper intramuscular positions, displaced radially from each other by about 40-70 microm. Two morphologically distinct types of ICs were found among enzymatically dispersed cells from this vessel: small multipolar cells with stellate-shaped bodies not exceeding 20 microm, and spindle-shaped cells from 40 to 300 microm in length with numerous branching processes. Relaxed smooth muscle cells (SMCs) had a more constant length (90-150 microm). The cell membrane capacitance was 46.5+/-2.2 pF in SMCs, 39.7+/-2.4 pF in spindle-shaped ICs and 27.8+/-0.7 pF in multipolar ICs. Although darker under phase contrast, after loading with fluo-4 AM, single isolated ICs of both types usually had brighter fluorescence than SMCs and displayed various spontaneous calcium events, including Ca(2+) sparks and Ca(2+) waves. Ca(2+) waves were usually followed by contraction of SMCs but no change in shape of ICs. In some ICs spontaneous [Ca(2+)](i) transients (lasting about 2s) which propagated towards the end of the processes were observed. Physical contacts between the processes of ICs and the body of one or more SMCs survived the isolation procedure. Application of noradrenaline (1-10 microM), caffeine (1-10 mM) or high-K(+) solution (60mM) led to a rise of [Ca(2+)](i) in both SMCs and ICs evoking contraction of SMCs but not ICs. No differences in electrophysiological characteristics between single enzymatically isolated IC and SMC were detected; thus, the resting membrane potential estimated under current-clamp conditions was -46.5+/-2.0 mV in spindle-shaped ICs and -45.6+/-2.7 mV in SMCs. Under voltage-clamp, both ICs and SMCs revealed a well-developed voltage-gated nifedipine-sensitive L-type Ca(2+) current, a set of K(+) currents, including spontaneous transient outward currents (STOCs) but no Na(+) current. This study for the first time directly demonstrated the presence in vascular tissue of ICs. Possible roles for ICs including their involvement in spontaneous activity of the vessel were discussed.     

The effect of the composition of the medium on the concentration of free calcium ions in the cells of the follicular wall in the common frog and in the clawed toad]

The concentration of intracellular free calcium ions in the follicle wall cells and in the follicle cells of Rana temporaria in Ringer solution is 150 +/- 10 and in the follicle wall cells of Xenopus laevis, 220 +/- 10 nM. In a chloride-free saline, its concentration in the same cells is 2.5-3 times that in Ringer solution. Voltage-dependent Ca(2+)-channel blockers diltiazem and verapamil (100 microM) reduce the level of intracellular free calcium ions in R. temporaria follicle wall cells cultivated in a chloride-free saline to 170 +/- 20 nM, which practically does not differ from the level in Ringer solution. Inhibitors (100 microM) decrease the rate of "spontaneous" maturation of R. temporaria follicle-enclosed oocytes both in chloride-free and Ringer solutions. It was concluded that an increased level of intracellular free calcium ions in the follicle cells, among other factors, may determine the stimulating effect of the medium (Ringer or chloride-free solution) on "spontaneous" maturation of follicle-enclosed amphibian oocytes. Voltage-dependent calcium channels appear to be involved in Ca2+ influx into the cells.     

An Extended Bidomain Framework Incorporating Multiple Cell Types

The muscular layers within the walls of the gastrointestinal tract contain two distinct cell types, the interstitial cells of Cajal and smooth muscle cells, which together produce rhythmic depolarizations known as slow waves. The bidomain model of tissue-level electrical activity consists of single intracellular and extracellular domains separated by an intervening membrane at all points in space and is therefore unable to adequately describe the presence of two distinct cell types in its conventional form. Here, an extension to the bidomain framework is presented whereby multiple interconnected cell types can be incorporated. Although the derivation is focused on the interactions of the interstitial cells of Cajal and smooth muscle cells, the conceptual framework can be more generally applied. Simulations demonstrating the feasibility of the proposed model are also presented.     

Role of the Plasma Membrane Ca2+-ATPase Pump in the Regulation of Rhythm Generation by an Interstitial Cell of Cajal: A Computational Study

Neurophysiology - Gastrointestinal motility is based on the rhythmic activity of interstitial cells of Cajal (ICCs). The ICC rhythm generation relies upon characteristic Ca2+-handling mechanisms...     

Characterization of in vitro gutlike organ formed from mouse embryonic stem cells

Using an embryoid body (EB) culture system, we have made a functional organlike cluster: the "gut" from embryonic stem (ES) cells (ES gut). There are many types of ES clusters, because ES cells have a pluripotent ability to develop into a wide range of cell types. Before inducing specific differentiation by exogenously added factors, we characterized comprehensive physiological and morphological properties of ES guts. Each ES gut has a hemispherical (or cystic) structure and exhibits spontaneous contractions [mean frequency: 13.5 ± 8.8 cycles per min (cpm)]. A dense distribution of interstitial cells of Cajal (ICC) was identified by c-Kit immunoreactivity, and specific subcellular structures of ICC and smooth muscle cells were identified with electron microscopy. ICC frequently formed close contacts with the neighboring smooth muscle cells and occasionally formed gap junctions with other ICC. Widely propagating intracellular Ca²⁺ concentration oscillations were generated in the ES gut from the aggregates of c-Kit immunopositive cells. Plateau potentials, possibly pacemaker potentials in ICC, and electrical slow waves were recorded for the first time. These events were nifedipine insensitive, as in the mouse gut. Our present results indicate that the rhythmic pacemaker activity generated in ICC efficiently spreads to smooth muscle cells and drives spontaneous rhythmic contractions of the ES gut. The present characterization of physiological and morphological properties of ES gut paves the way for making appropriate models to investigate the origin of rhythmicity in the gut. intracellular calcium concentration oscillation; interstitial cells of Cajal; peristalsis     

Snapshots of mammary gland interstitial cells: methylene-blue vital staining and c-kit immunopositivity

We show here that methylene-blue supravital staining of specimens from normal human mammary gland reveals (selectively) interstitial (stromal) cells, with 2-3 long (20-80 microm), thin, moniliform processes. Such cells appear c-kit/CD117 positive, either by immunohistochemistry (IHC) or immunofluorescence (IF). Since these features (affinity for methylene blue, c-kit positivity, and characteristic processes) define archetypal interstitial cells of Cajal (ICC) in light microscopy, our results suggest the existence of Cajal-like cells in the interstitium of human normal mammary gland.     

Cytofluorometry of electromagnetically controlled cell dedifferentiation.

Cellular morphology changes, which appear related to dedifferentiation (despecialization), have been produced in vitro in the nucleated red blood cell of the frog. This has been achieved by controlled alterations in the electrochemical environment of these living cells, both by a selective modification of the ionic concentrations of an isotonic amphibian Ringer solution, and by the electromagnetic induction of pulsating current having specific waveform parameters. Laser flow microfluorometry shows that the modified Ringer solution is able, per se, to partially trigger the process in the same time interval that certain induced current waveforms can significantly affect the number of cells in the so-called dedifferentiated state. It has also been found that, for a given waveform, the repetition rate appears to have a significant effect on the rate of cell change. Preliminary automated image analysis of cell smears suggests that dedifferentiated and normal cells have the same integrated optical density but different nuclear areas. In conclusion, it appears that, after the initial electrochemical trigger, the early stage of the process, when the cells move from a state of specialized function to one of less specific activity, is the unfolding of their chromatin supercoil, not involving DNA synthesis. Then cytofluorometry allowed us to identify, for the first time, fundamental modifications which occur in the cell nucleus under electromagnetic exposure.     

Influence of culture medium osmolality on maturation and ovulation of Rana temporaria oocytes stimulated in vitro by the pituitary gland extract or progesterone

The influence of diluted Ringer solution on ovulation and maturation of common frog oocytes stimulated in vitro by homologous pituitary extract (0.005 pit/ml) or progesterone (1 pg/ml) was studied. During wintering, the dilution of Ringer solution led to a decreased percentage of oocytes ovulated and matured under the influence of both inducers. As the season of reproduction approached, the dependence of oocyte maturation and ovulation on the Ringer solution dilution weakened. Possible causes of different dependence of the ovulation of amphibian and sturgeon oocytes stimulated by gonadotropic hormones or progesterone on the culture medium osmolality is discussed.     

Influence of culture medium osmolality on maturation and ovulation of common frog oocytes stimulated in vitro by pituitary extract or progesterone

The influence of diluted Ringer solution on ovulation and maturation of common frog oocytes stimulated in vitro by homologous pituitary extract (0.005 pit/ml) or progesterone (1 μg/ml) was studied. During hibernation, the dilution of Ringer solution led to a decreased percentage of oocytes ovulated and matured under the influence of both inducers. As the season of reproduction approached, the dependence of oocyte maturation and ovulation on the Ringer solution dilution was reduced. Possible causes of different dependence of the ovulation of amphibian and sturgeon oocytes stimulated by gonadotropic hormones or progesterone on the culture medium osmolality is discussed.     

Participation of Na, K-ATPase in the mechanism of isotonic fluid absorption by the epithelium of the frog gallbladder]

In experiments on the isolated frog gallbladders it was shown that addition of ouabain (1.10(-4) M) or noradrenaline 3.10(-6) M) into the incubation Ringer solution from the serous surface of the gallbladder and also replacement of K+ in the solution for the equivalent quantity of Na+ ions caused a reduction of the absorption of isotonic fluid by the epithelium and a fall of the Na,K-ATPase activity in its cells. Noradrenaline also cased a reduction of Mg-ATPase activity. A significant positive correlation was found between the transport rate of the isotonic fluid by the epithelium and the Na,K-ATPase activity in its cells.     

Repolarization of striated muscle fibers by the antifatigue agent,K-Mg-aspartate

Resting membrane potentials of isolated frog sartorius muscles were measured under a variety of conditions using intracellular glass microelectrodes. Muscle cells depolarized by the addition of 5.0 or 10.0 mM KCl to the bathing Ringer solution can be repolarized some 5 to 10 mV by the substitution of an equivalent amount of K-aspartate for KCl in the presence of 2.0 mM Mg⁺⁺. The repolarization produced by this method persists when the muscle is again placed in the initial KCl solution, thus eliminating the possibility that the hyperpolarization is due to the reduction of chloride in the bathing medium. If for some reason the resting membrane potential of the muscle fibers is considerably below (less negative than) the normal level of 92 mV reported for muscles bathed in 2.5 mM Ringer solution, the substitution of 2.5 mM K-aspartate for the 2.5 mM KCl and the addition of 2.0 mM Mg-aspartate to the Ringer solution will, within 15 minutes, repolarize the fiber to the normal level. Magnesium ions alone will not produce the observed repolarization nor can it be attributed to a reduction in the activity of the potassium in the Ringer solution.     

Spontaneous Rhythmic Inward Currents Recorded in Interstitial Cells of Rabbit Portal Vein

It is now well established that smooth muscle of the portal vein exhibits spontaneous rhythmic contraction in vitro. The present study was designed to investigate the pacemaking mechanism(s) underlying the spontaneous rhythmic contractions in the rabbit portal vein (RPV). Using whole-cell patch clamp techniques, spontaneous inward currents were recorded at ?60 mV of holding potential in freshly dispersed c-Kit immunopositive interstitial cells (ICs) isolated from sections of RPV. The inward currents were abolished by caffeine, FCCP, thapsigargin, and ryanodine, but were partially inhibited by 2-APB. Both gadolinium, a non-selective cation channel inhibitor, and niflumic acid, a chloride channel blocker, inhibited the inward currents completely. Replacement of external Na⁺ with NMDG⁺ also blocked the inward currents. W-7, a calmodulin inhibitor, increased both the amplitude and frequency of the inward currents. Taken together, these results indicate that non-selective cationic channels are involved in the generation of spontaneous inward currents recorded from ICs. Intracellular calcium concentration and calmodulin regulate the spontaneous inward currents, which may account for spontaneous rhythmic contraction in the RPV, but a role of chloride channels may not be excluded in the present study.     

The Role of Calcium Ions in Modulation of Impulse Activity of the Isolated Muscle Spindle of the Frog Rana temporaria

The effect of various concentrations of Ca⁺² and Mg⁺² as well as of calcium channel blockers verapamil and nifedipin on impulse activity of frog isolated muscle spindles was studied. Removal of Ca ions from the external Ringer solution was established to increase spontaneous and evoked activity of the muscle spindle. A 4- and 8-fold increase of Ca⁺² concentration produces inhibition and complete cessation of the spontaneous and evoked activity in the muscle spindle. Replacement of Ca⁺² by Mg⁺² is observed to cause no statistically significant change of the spontaneous activity of the isolated muscle spindle; at the same time, at the dynamic spindle extension, the impulse activity rate at the dynamic and static phases of the response rises. Nifedipin and verapamil, blockers of Ca⁺² channels, suppress impulse activity both in norm and on the background of increased impulse activity evoked by removal of Ca⁺² from the external solution. An increase of muscle spindle impulse activity after the removal of Ca⁺² from the external solution is accounted for by transformation of calcium channels of the muscle spindle sensory endings into selective sodium channels.     

Arterial perfusion of frog tongue for intracellular recording of taste cell receptor potential

The frog tongue was perfused through its artery with a Ringer solution using a peristaltic pump, and a method was developed to record stable intracellular receptor potentials of taste cells. Perfusing at 0.05 ml/min with a Ringer solution containing 5% dextran did not cause tongue edema, but perfusing at the same rate with Ringer without dextran caused edema. After perfusion at 0.05 ml/min with 100 mM K Ringer, the membrane potential of taste cells gradually decreased and reached a constant level in about 30 min, indicating that the intercellular fluid of the tongue could be replaced within this time period. While the artery of the frog tongue was perfused at 0.05 ml/min with Ringer containing 5% dextran, intracellular receptor potentials of taste cells elicited by four basic taste stimuli (1 M NaCl, 10 mM quinine-HCl (Q-HCl), 1 mM acetic acid and 1 M galactose) were similar to those obtained from the control taste cells under normal blood flow.     

Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium

Inward-rectifier K channel: using macroscopic voltage clamp and single- channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current- voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell- attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. Conclusions: under physiological ionic gradients, a 15-pS inward- rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.     

C-kit immunopositive interstitial cells (Cajal-type) in human myometrium

Previous reports describing Cajal-like interstitial cells in human uterus are contradictory in terms of c-kit immunoreactivity: either negative (but vimentin-positive) in pregnant myometrium, or positive, presumably in the endometrium. The aim of this study was to verify the existence of human myometrial Cajal-like interstitial cells (m-CLIC). Six different, complementary approaches were used: 1) methylene-blue supravital staining of tissue samples (cryosections), 2) methylene blue and Janus green B vital staining (m-CLIC and mitochondrial markers, respectively), and 3) extracellular single-unit electrophysiological recordings in cell cultures, 4) non-conventional light microscopy on glutaraldehyde/osmium fixed, Epon-embedded semi-thin sections (less than 1 microm) stained with toluidine blue (TSM), 5) transmission electron microscopy (TEM), and 6) immunofluorescence (IF). We found m-CLIC in myometrial cryosections and in cell cultures. In vitro, m-CLIC represented approximately 7% of the total cell number. m-CLIC had 2-3 characteristic processes which were very long (approximately 60 microm), very thin (< or =0.5 microm) and moniliform. The dilated portions of processes usually accommodated mitochondria. In vitro, m-CLIC exhibited spontaneous electrical activity (62.4+/-7.22 mV membrane potentials, short duration: 1.197+/-0.04 ms). Moreover, m-CLIC fulfilled the usual TEM criteria, the so-called 'gold' or 'platinum' standards (e.g. the presence of discontinuous basal lamina, caveolae, endoplasmic reticulum, and close contacts between each other, with myocytes, nerve fibers and/or capillaries etc.). IF showed that m-CLIC express CD117/c-kit, sometimes associated with CD34, with vimentin along their processes. In conclusion, we describe myometrial Cajal-like interstitial cells that have affinity for methylene blue and Janus green B vital dyes, fulfill (all) TEM criteria, express CD117/c-kit and have spontaneous electric activity.