Non-enzymatic isolation and culture of channel catfish hepatocytes.

An alternative method has been developed for isolating and culturing hepatocytes from livers of channel catfish. Hepatocytes are prepared using a collagenase-free perfusion system that relies on the chelating properties of ethylenediamine tetraacetic acid (EDTA). Hepatocyte yields of up to 3.6 x 10(8) cells per 100 g body weight have been achieved with initial viabilities routinely exceeding 95%. Cells isolated by this method and incubated in osmotically corrected culture medium at physiological pH have been maintained for several weeks in culture with minimal cell loss. During the first 24-48 h of culture, hepatocytes begin to link together and show structures that closely resemble those seen in intact liver (e.g. bile canaliculi, sinusoids). Cells cultured at 15 degrees C for 7 days maintain levels of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and lactate dehydrogenase (LDH), activity similar to those measured in vivo.

Seasonal variations in the temperature acclimation response of the channel catfish, Ictalurus punctatus.

Channel catfish were collected on 11 different dates from October 1991 to July 1993 and acclimated in the laboratory to 7 degrees C, 15 degrees C, or 25 degrees C for 6 wk. Hepatosomatic index, mg protein mg-1 DNA, total liver DNA and protein, and the activities of liver glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, lactate dehydrogenase, and malate dehydrogenase were measured to examine seasonal variation in the acclimation response. Liver and muscle cytochrome oxidase and lactate dehydrogenase activities were measured to compare tissue-specific responses. Hepatosomatic indexes of fall and winter channel catfish were highest at 7 degrees C, with values at 15 degrees C higher than at 25 degrees C, while spring and summer fish had the highest values at 15 degrees C, with values at 7 degrees C higher than those at 25 degrees C. Acclimation patterns for total liver protein and DNA, mg protein mg-1 DNA, and glycogen were generally higher in cold temperatures but varied seasonally in an unpredictable manner. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malate dehydrogenase demonstrated positive acclimation in the fall and winter; fish collected in the spring and summer showed little or inverse acclimation. Liver lactate dehydrogenase activity showed little or no positive compensation at any time of the year. Cytochrome oxidase activity showed positive acclimation in muscle but not liver. All liver enzymes, even those that showed marginal acclimation on a protein basis, showed positive acclimation when activity was expressed on a whole-liver basis.

Rhythmic electrical and mechanical activity in stomach of toad and frog.

Rhythmic slow spikes and contractions at 2-5/min occur in frog/toad stomach when longitudinal muscle is present, not in the circular layer after removal of the longitudinal layer. Interstitial cells of Cajal occur in frog stomach in the longitudinal layer and in toad stomach mainly in the longitudinal layer, with some between layers. They were identified by staining with methylene blue, the fluorescent dye rhodamine 123, toluidine blue, and by electron microscopy. Interstitial cells resemble in ultrastructure those observed in mammalian intestine by small cell diameter, long processes forming a network, large nuclei, mitochondria, caveolae, endoplasmic reticulum, and absence of fibrillae. Rhythmic activity in stomachs stained with methylene blue is abolished by bright illumination. Rhythmic slow spikes and contractions are reduced and frequency is lowered when Ca2+ concentration in the medium is significantly decreased; in high Ca2+ these functions are increased. Threshold concentration of Ca2+ for maintaining slow spikes was between 10(-7) and 10(-6) M Ca2+. Ba2+ and 2.5 mM increases slow spike frequency and amplitude; Ba2+ increases contractions proportionately more than spikes. Slow spikes are reduced or blocked by verapamil, nifedipine, and Cd2+ and are enhanced by BAY K 8644. Quaternary ammonium compounds tetraethylammonium and tetrapentyl-ammonium bromide prolong slow spikes and enhance contractions. Replacement of NaCl by LiCl, methyl glutamine, or treatment with amiloride reduces slow spike amplitude and to a lesser extent frequency. This indicates efflux of Ca2+ by Na+/Ca2+ exchange. Contractions, but not spikes, are reduced by the calmodulin blocker trifluoroperazine. Contractions are enhanced, but slow spikes are not altered by ouabain. This suggests retention of Ca2+ when a tonic Na+/K+ pump is blocked. A model for rhythmicity includes influx of Ca2+ via L-channels, repolarization by IKCa2+, efflux of Ca2+ by Na+/Ca2+ exchange, and efflux of Na+ by a Na+/K+ pump. Frequency is determined by rate constants for both influx and efflux of Ca2+.

Diversity of rhythmicity in gastrointestinal smooth muscles.

Diversity in smooth muscles is well shown by differences in gastrointestinal rhythms. Basic rhythmic activity apparently occurs only when there is interaction between clusters of cells. It is postulated that the oscillations of basic electrical rhythms result from action of a membrane ionic network. In addition to slow waves, gastrointestinal muscle is capable of acetylcholine-induced rhythms, of spontaneous "slow spikes" that may be similar to the Ca2+ component in stomach, of neurally paced migrating complexes, of Ca-EGTA plateaus and of stretch induced rhythms.

Immunocytochemistry of the interstitial cells of Cajal in the rat intestine.

There is experimental evidence suggesting that the interstitial cells of Cajal are essential for rhythmic slow waves of the smooth muscle layers of the mammalian small intestine. Different investigators have identified them variously as modified neurons, glia, fibroblasts or modified smooth muscle cells. Since histological categorization bears on understanding their function, we have examined the immunoreactivity of the myenteric plexus of the rat small intestine, paying special attention to the cell type identified as Thuneberg's Type I-ICC. Polyclonal and monoclonal antisera directed against 4 intermediate filament proteins: neurofilament protein, glial fibrillary acidic protein, vimentin and desmin were used. In addition, antisera directed against neuron-specific enolase, substance P and vasoactive intestinal polypeptide were also tested for reactivity. Type I-ICCs were immunonegative to all the antisera directed against intermediate filament proteins and neuropeptides. However, some Type I-ICCs were immunopositive to antisera against neuron-specific enolase. On the basis of these results and the distribution of immunoreactivities to these kinds of antisera in other tissues, we suggest that Type I-ICCs are distinct from typical myenteric neurons, from glia, from fibroblasts and from smooth muscle fibers. Staining with antiserum against neuron-specific enolase suggests a relation to some type of neuron.

Comparative physiology and biochemistry: challenges for the future.

1. Comparative physiology is distinguished from other types of physiology by treating the diversity of solutions of functional problems and by using kind of animal as a functional variable. 2. The strength of comparative physiology os its capacity to give some solutions to problems in basic biology. 3. Specific examples of subject areas to which comparative physiology contributes are: (a) mechanisms underlying evolution; (b) the nature of speciation; (c) comparative cognitive science, neural models for behavior; and (d) applications of molecular techniques to physiology of whole animals. 4. Applications continue in ecology, medicine and agriculture. 5. The breadth of the comparative approach to physiology has important philosophical implications.

Cold-acclimation-induced protein hypertrophy in channel catfish and green sunfish.

1. Following acclimation of channel catfish to a reduction in temperature from 25 degrees to 15 degrees C, there were approximately two-fold increases in liver mass, cell size, total protein, and total enzyme activity, relative to activity per milligram of protein and per gram wet weight of tissue, indicating tissue hypertrophy. There was no change in either total liver DNA content or protein concentration per gram weight. 2. Green sunfish, unlike catfish, showed virtually no change in liver mass following cold acclimation. However, sunfish showed a net increase in total liver protein content and an increase in protein concentration. The increase in protein content was balanced by a reciprocal and equivalent decrease in glycogen content. Consequently, liver mass was maintained. 3. During cold acclimation both catfish and sunfish showed an increase in ventricular heart mass and protein content, but no change in protein concentration. 4. The activities of several enzymes were measured in liver from 15 degrees C and 25 degrees C steady-state-acclimated catfish and at intervals following transfer from 15 degrees to 25 degrees C and from 25 degrees to 15 degrees C. Total tissue enzyme activity showed positive compensation which correlated with the change in liver mass and protein content. Specific activities based on protein and on wet weight showed dissimilar acclimatory patterns. Two enzymes - cytochrome oxidase and lactate dehydrogenase - showed inverse compensation in specific activity but positive compensation in total activity. Citrate synthase, glucose-6-phosphate-dehydrogenase and 6-phosphogluconate dehydrogenase showed positive compensation in both specific and total activities. 5. The increase in tissue protein content or 'protein hypertrophy' occurred with cell hypertrophy in cold-acclimated catfish, while protein hypertrophy occurred as an increased protein concentration without cell hypertrophy in sunfish. This phenomenon is considered adaptive in that it permits a compensatory increase in the total enzymatic capacity of a tissue. The two-fold increases in total enzyme activities, superimposed on either an increase or decrease in specific activity, suggest that two biochemical mechanisms may be operative during cold-induced liver hypertrophy, one effecting a specific step in protein translation at a point common to the synthesis of all proteins and a second targetted pretranslationally, i.e., transcriptional regulation.

Two types of 'slow waves' in intestinal smooth muscle of cat.

1. Smooth muscle from cat small intestine shows two types of spontaneous slow electrical waves in the frequency range of 10-15 min-1. One type of slow wave is a ouabain-sensitive, atropine-insensitive spontaneous oscillation. The other type of wave can be induced by acetylcholine (ACh), is blocked by atropine, and is not blocked by ouabain. 2. Ouabain-sensitive slow waves rise directly from the baseline, are near sinusoidal and may or may not have spikes. ACh-induced waves have pre-potentials, are usually topped by spikes and show after-hyperpolarization. 3. The two types of rhythmic wave differ in ionic and metabolic requirements and drug sensitivity. Ouabain-sensitive waves occur only in intestinal muscle attached to a boundary layer containing interstitial cells; ACh-induced waves can occur in strips of muscle lacking boundary cells. 4. Na+ pump inhibitors ouabain, cold and K+-free solution, reduce amplitude but not frequency of ouabain-sensitive slow waves. 5. The ACh-induced waves require higher extracellular concentrations of Na+ and Ca2+ and can occur in preparations in Li+-Krebs solution; the ouabain-sensitive rhythm persists in lower concentrations of Na+ and Ca2+ and is not supported by Li+. The ouabain-sensitive waves are more sensitive to cyanide and less sensitive to cooling than the ACh-induced waves. 6. Guinea-pig intestine shows only one type of rhythmic wave, which is atropine sensitive and resembles in shape the ACh-induced wave of other species. Ouabain increases the frequency of the guinea-pig rhythm. 7. It is concluded that intestinal muscle of most mammals, but not of guinea-pig, is capable of two types of slow electrical rhythms.

Boundary cells between longitudinal and circular layers: essential for electrical slow waves in cat intestine.

Electrical slow waves were recorded by intracellular electrodes and by quasi-intracellular pressure and suction electrodes from muscle fibers at different levels in edgewise preparations of cat jejunum. Simultaneous recordings from longitudinal and circular muscle layers showed similar resting potentials from either muscle layer near the boundary zone, and lower resting potentials in cells of circular muscle near the submucosa. Slow waves were maximal in amplitude at the boundary between the two layers and spread electrotonically away from the boundary in both layers. Bipolar recordings were of opposite polarity on the two sides of the boundary. Amplitudes of slow waves from inner circular fibers were significantly lower than from outer circular fibers. Small strips of each muscle layer were prepared with or without the attached interstitial cells of Cajal plexus as identified by methylene blue staining. Either muscle layer showed slow waves from regions where interstitial cells of Cajal (ICC) were observed after the recording. No slow waves were recorded from either layer from regions where ICC were not observed. Strips containing ICC but not strips lacking ICC could be driven electrically. Since blocking of neurons does not abolish slow waves and since regions of muscle lacking ICC do not have slow waves, it is concluded that the interstitial cells (ICC-I) are most likely the boundary elements essential for slow waves in either layer of intestinal muscle.

Waxing and waning of slow waves in intestinal musculature.

Electrical slow waves from cat or rabbit small intestine show more variability when recorded in vivo than in vitro. One pattern of variation is waxing and waning of amplitude, or "spindling," during which two rhythms of slightly different frequency come in and out of phase. Fourier power analyses of slow waves during spindles show two frequency peaks of slow waves differing by 0.4-5.0 waves/min and corresponding to measured spindle durations of 12-150 s. Spindles can be induced in vitro in rabbit intestine by K depolarization of approximately 15 mV. Histograms of intracellular recordings of slow nonspindling waves show variation of 0.5-1.0 s on either side of a mean slow wave duration. Spindles are abolished by treatments that reduce electrical coupling between cells, e.g., hypertonic sucrose or lowered pH, but changes in calcium do not alter spindles. Simultaneous recordings by two electrodes in the longitudinal axis show synchrony of spindles at 2- to 3-mm but not at 5-mm separation and synchrony circumferentially to the opposite side of a segment. Contractions, both in vivo and in vitro, correspond with electrical spindles in amplitude. Spindle durations were significantly shorter in vivo than in vitro, indicating a significantly greater difference in vivo in the competing frequencies at the point of recording (P less than 0.01). Three conditions favoring waxing and waning are slight depolarization, variation in slow wave frequency at a point, and electrotonic coupling between muscle fibers. Spindles provide for rhythms of contractions of a 1- to 2-min period.

The effects of temperature acclimation on the resting membrane of skeletal muscle fibres from green sunfish.

Conductive properties of muscle fibres from green sunfish (Lepomis cyanellus) acclimated to different temperatures were examined. The relative membrane permeability to chloride and potassium ions, PCl/PK, measured at acclimation temperature, was approximately 7.0 after acclimation at 25 degrees C and 1.3 after acclimation at 7 degrees C. This difference was due to a six-fold reduction in the membrane chloride conductance upon acclimation to 7 degrees C as compared to 25 degrees C-acclimated fibres. Mean (+/- S.E.M.) values of the chloride conductance were 554 +/- 68 microseconds cm-2 in warm-acclimated sunfish, and 75 +/- 9 microseconds cm-2 in cold-acclimated sunfish. Membrane capacitance also differed significantly between the two acclimation groups. When the temperature was varied acutely, the magnitude of the chloride conductance exhibited a maximum Q10 of only 1.9, compared with a Q10 of 3.0 associated with acclimation. Upon transferring 25 degrees C-acclimated sunfish to holding tanks at 7 degrees C, the total membrane resistance exhibited a sigmoidal increase over about 14 days, and a steady membrane capacitance was achieved in about 10 days. For 7 degrees C-acclimated sunfish, transferred to 25 degrees C, resistance showed a sigmoidal decrease over 10 days and capacitance was steady after 8 days. The results indicate that thermal acclimation of the muscle membrane involves cellular regulatory processes which underlie significant changes in the electrical properties of the fibre.

Effects of alterations in calcium levels on cat small intestinal slow waves.

Intact segments of cat intestinal muscle and strips of isolated longitudinal muscle were treated with agents that reduce intracellular calcium concentration: incubation in 0-calcium saline, treatment with calcium conductance blockers, elevated extracellular magnesium concentration, or alkalinization with NH4Cl. These treatments reduced amplitude and frequency of slow waves in intact segments but only reduced frequency in isolated longitudinal muscle. The reduction in frequency was characterized by prolongation of the hyperpolarized phase of the slow waves. Treatments that would moderately increase intracellular calcium concentration, i.e., increasing external calcium to four times normal levels or lowering pH by CO2, increased slow-wave frequency. Increased frequency was associated with reduced amplitude and shortening of the hyperpolarized phase of the slow waves. Greater than four times normal calcium levels and intense spiking reduced slow-wave frequency. Chlorotetracycline fluorescence, an indicator of intracellular calcium concentration, showed fluctuations synchronous with slow waves. It is concluded that the reactions that pace the generation of slow waves are dependent on the level of intracellular calcium.

Variable homeoviscous responses of different brain membranes of thermally-acclimated goldfish.

The effects of thermal acclimation of goldfish upon the bulk fluidity of synaptic, mitochondrial and myelin membrane fractions of brain was determined using steady-state and differential polarised phase fluorimetry. Membrane fluidity decreased in the order, mitochondria greater than synaptic membranes greater than myelin. in each case membranes from cold-acclimated goldfish were more fluid than the corresponding membranes of warm-acclimated goldfish, though the adjustment of fluidity in each case was insufficient to compensate for the direct effects of the temperature difference. The extent of fluidity compensation was greatest in the mitochondrial fraction and least in the myelin fraction, indicating heterogeneous responses in different membrane-types. Steady-state and dynamic fluorimetric techniques provided identical estimates of the homeoviscous responses, indicating that despite its short-comings, the steady-state technique provided as good a measure of adaptive responses as the more complex and sophisticated technique.

Depolarization-induced contractile activity of smooth muscle in calcium-free solution.

In calcium-free solution, strips of cat intestinal muscle developed slow, rhythmic electrical potential changes that triggered contractions. Some strips failed to develop spontaneous electrical activity in calcium-free solution but responded with contractions to depolarization by direct electrical stimulation or by treatment with barium chloride, potassium chloride, or acetylcholine. Similar results were obtained with segments of cat stomach, colon, esophagus, bladder, uterus, and vena cava, as well as with rabbit vena cava. In calcium-free saline, rat small intestinal muscle showed fast electrical activity with accompanying development of a tetanuslike contraction. After 60 min in calcium-free solution, cat small intestinal muscle retained 17.7% of its original concentration of calcium. It is concluded that in some smooth muscles, depolarization-triggered release of intracellular calcium does not require an associated influx of calcium.

Temperature-sensitive neurons in the preoptic region of sunfish.

Single-unit, extracellular recordings were made from spontaneously active, thermosensitive neurons in the preoptic region of green sunfish acclimated to 25 degrees C. Activity of single cells was monitored during increases and decreases in local brain temperature over approximately a 10 degrees C range. Deep-body and skin temperatures were maintained at 25 degrees C. Of 276 neurons, 81% were insensitive, 17% were warm sensitive, and 2% cold sensitive. Warm responses were grouped into three basic types: exponential, linear, and nonlinear. All cold-sensitive neurons responded in a similar nonlinear manner. Mean levels of firing rate of thermosensitive neurons at 25 degrees C brain temperature ranged from 6 +/- 1.1 impulses/s to 22.7 +/- 10.8 impulses/s. Thermosensitivities were as high as 5.2 +/- 0.9 impulses . -1 . degrees C-1. Anatomic location of these neurons within the preoptic region appears random with some trend for the exponentially responding, warm-sensitive neurons to be located more medial than the other thermosensitive cell types. A small number of neurons were located in the ventrolateral telencephalon. In general, the thermosensitive responses observed resemble those found in other ectotherms and mammals with some exceptions.

Intracellular recordings from thermosensitive preoptic neurons.

Intracellular recordings were made from locally thermosensitive preoptic neurons in the green sunfish, Lepomis cyanellus. Stable resting potentials, action potentials, and spontaneous synaptic activity were observed over approximately 4 degrees to 5 degrees C changes in local brain temperature. A small percentage of the warm-sensitive neurons showed exponential firing-rate responses to temperature. These cells discharged rhythmically, lacked visible synaptic input, and showed slowly depolarizing potentials leading to action potentials. Other linear and nonlinear warm-sensitive and cold-sensitive neurons showed spontaneous excitatory and inhibitory synaptic potentials giving rise to action potentials. Cells that appear to be endogenously active may be true thermodetectors, and other thermosensitive neuronal activity may be synaptically mediated.