Segmental heterogeneity of the rat colon in the response to activators of secretion on the cAMP-, the cGMP- and the Ca2+-pathway

The electrolyte transport was compared in proximal and distal segments of the rat colon under control conditions and after induction of secretion on the CAMP-, the cGMP- and the Ca²⁺-pathway. Baseline short-circuit current was decreased by indometacin and tetrodotoxin in the distal colon, indicating a spontaneous production of neuronally acting prostaglandins. In contrast, baseline short-circuit current in the proximal colon was decreased only by indometacin, but not by tetrodotoxin. Unidirectional flux measurements revealed that in the distal colon sodium and chloride were absorbed, while the proximal colon secreted chloride. A morphological comparison between the distal and proximal epithelium revealed that the zonulae occludentes and the microvilli were longer in the distal colon. The size of the Golgi apparatus was several times larger in the crypt than in the surface region without differences between proximal and distal colon. Distal segments were more sensitive to an activator of the Ca²⁺-pathway, carbachol, or activators of the CAMP-pathway such as forskolin and a CAMP-analogue. In contrast, the activation of the cGMP-pathway by a cGMP-analogue or by the heat-stable enterotoxin of E. coli (STa) was more effective in the proximal colon. The results give evidence for a segmental specificity with regard to the intracellular pathways responsible for the activation of secretion.     

Segmental heterogeneity of rat colonic electrogenic secretion in response to the bacterial enterotoxin Escherichia coli STa in vitro.

The effects of the bacterial toxin Escherichia coli STa on electrogenic secretion in proximal, mid and distal rat colon were examined in vitro using the short-circuit current (Isc) as an index of secretion. All areas showed toxin-elevated Isc which was reduced by bumetanide. The proximal colon was the most sensitive (lowest ED50) but displayed the lowest maximal increase in Isc, the mid colon had the largest maximal electrogenic response while the distal colon proved to be the least sensitive (highest ED50). Atropine (1 microM) and tetrodotoxin (1.25 microM) reduced the maximum Isc response to STa by up to 42%, indicating that the enteric nervous system may be partly involved in the response.     

Acceleration of Ca(2+) repletion in the junctional sarcoplasmic reticulum and alternation of the Ca(2+)-induced Ca(2+)-release mechanism in hypertensive rat (SHR) cardiac muscle

We estimated the time taken for a repletion of the junctional sarcoplasmic reticulum (JSR) Ca(2+) stores from a family of mechanical restitution curves after twitches of various magnitudes in the cardiac muscle of hypertensive rats (SHR), using a method described previously (Tameyasu et al. Jpn J Physiol. 2004;54:209-19), to evaluate abnormality in Ca(2+) handling by cardiac JSR in hypertension. We found no differences in contractility or in the time course of mechanical restitution between SHR and the controls (WKY) at 3 weeks of age. In comparison to WKY, 7- and 20-week-old SHR showed a greater rested state contraction (RST) and similar or smaller rapid cooling contracture, suggesting that their JSR contains a similar amount of Ca(2+) at saturation, but releases more Ca(2+) upon stimulation. The adult SHR and WKY showed similar mechanical restitution time courses, but the adults had longer pretwitch latencies. The function G(t) representing the time course of JSR Ca(2+) store repletion in adult SHR exceeded the WKY value at t < or = 0.5 s, but the function H(t) representing JSR [Ca(2+)] change corresponding to the mechanical restitution after RST was smaller in the adult SHR at t < or = 0.5 s, resulting in smaller H(t)/G(t) in adult SHR at t < or = 0.5 s. Deviations of G(t), H(t), and H(t)/G(t) from WKY were greater at 20 weeks than at 7. The results suggest an acceleration of JSR Ca(2+) store repletion and an alternation of the Ca(2+)-induced release of Ca(2+ )from the JSR in young adult SHR.     

Ryanodine receptors and the mediation of Ca2+-dependent anion secretion across rat colon

The properties of the Na(+)-Ca(2+) exchanger in isolated crypts from rat colon were studied using the Fura-2 imaging technique. The transport mode of the exchanger was reversed by replacing extracellular Na(+) by the impermeable cation, N-methyl-D-glucamine (NMDG(+)), so that the transporter mediated a Ca(2+) influx into the cells. Depletion of intracellular Ca(2+) stores by inhibitors of sarcoplasmatic endoplasmatic calcium ATPases (SERCA), i.e., cyclopiazonic acid (10(-5) mol l(-1)) or thapsigargin (10(-6) mol l(-1)), reduced the increase in [Ca(2+)](i) evoked by superfusion with NMDG(+), suggesting a cross-talk between the exchanger and the Ca(2+) stores. However, measurement of Ca(2+) influx with the Mn(2+) quench technique revealed that the activity of the exchanger was independent of the filling state of the stores. Instead, the obvious inhibition of the [Ca(2+)](i)response by SERCA blockers was due to a reduction of Ca(2+)-induced Ca(2+) release after inhibition of store-refilling. The functional presence of ryanodine receptors was demonstrated by the increase in [Ca(2+)](i)evoked by ryanodine (10(-7) to 3x10(-4) mol l(-1)) in a concentration-dependent manner. This effect was mimicked by cADP ribose (10(-5) mol l(-1)) in crypts permeabilized with saponin (10 mg l(-1)). Ruthenium red (5x10(-5) mol l(-1)) or high concentrations of ryanodine (3x10(-4) mol l(-1)) inhibited this response. In Ussing chamber experiments ruthenium red (5x10(-4) mol l(-1)) or a high concentration of ryanodine (10(-3) mol l(-1)) inhibited the increase in short-circuit current evoked by the cholinergic agonist, carbachol (5x10(-5) mol l(-1)). Consequently, Ca(2+)-induced Ca(2+) release may act as kind of amplifier during Ca(2+)-dependent Cl(-) secretion in order to maintain a long-lasting increase in the intracellular Ca(2+) concentration.     

Ca(2+)-dependent negative control mechanism for Ca(2+)-induced Ca2+ release in crayfish muscle.

The mechanism of termination of Ca(2+)-induced Ca2+ release (CICR) from the sarcoplasmic reticulum has been investigated in voltage clamped cut crayfish muscle fibres loaded with rhod-2. During depolarizing steps evoking calcium current (ICa), Ca2+ release was first activated. Then the release rapidly (tau approximately 6 ms) declined, as evidenced by the rate of change of the intracellular fluorescence signal representing a Ca2+ transient. The rapid termination of release was not accounted for by inactivation of the trigger ICa or depletion of Ca2+ from the SR, since the rate at which release declined was constant under conditions where the rate of ICa inactivation and the amount of Ca2+ released varied widely. Pre-elevations of [Ca2+]i with prepulses or photolysis of caged Ca2+ caused depression of Ca2+ release during a subsequent test pulse. When the rate of ICa onset was varied by applying voltage ramps with different slopes, currents with fast onset elicited larger Ca2+ release than calcium currents with slower onset, even though the amplitude of the currents was the same. These results suggest that a Ca(2+)-dependent negative control mechanism exists which mediates the termination of CICR independently of the duration of the trigger ICa and before significant depletion of Ca2+ in the SR occurs.     

The secretory response of the rat colon to the flavonol quercetin is dependent on Ca2+-calmodulin

The dietary flavonol quercetin induces chloride secretion in rat intestine. To clarify the underlying mechanisms, experiments were performed in Ussing chambers with tissue from rat proximal and distal colon. Quercetin induced an increase in short-circuit current (Isc), which was largely independent of submucosal neurons, as it was not affected by the neurotoxin tetrodotoxin. The effect of quercetin was blocked by the calmodulin antagonists trifluoperazine and ophiobolin A and was diminished by a blocker of Ca2+ release from intracellular stores (TMB-8), whereas the muscarinic receptor antagonist atropine was ineffective. The quercetin-induced Isc was abolished in Ca2+-free solution. The flavonol was able to further increase Isc after maximal stimulation of the cAMP pathway by forskolin. The Isc increase by the flavonol was differently affected by two analogous phosphodiesterase inhibitors. Whereas 3-isobutyl-1-methylxanthine (IBMX) antagonized the effect of quercetin, 8-methoxymethyl-IBMX had no effect. Both phosphodiesterase inhibitors similarly influenced the Isc increase induced by forskolin. These results indicate that the chloride secretion induced by quercetin in rat colon depends on Ca2+ and calmodulin. The cAMP pathway and inhibition of phosphodiesterase appear not to be responsible for the secretory activity of the flavonol.     

Ca(2+)-independent and Ca(2+)-dependent stimulation of quantal neurosecretion in avian ciliary ganglion neurons.

1. Although it is generally agreed that Ca2+ couples depolarization to the release of neurotransmitters, hypertonic saline and ethanol (ETOH) evoke neurosecretion independent of extracellular Ca2+. One possible explanation is that these agents release Ca2+ from an intracellular store that then stimulates Ca(2+)-dependent neurosecretion. An alternative explanation is that these agents act independently of Ca2+. 2. This work extends previous observations on the action of ETOH and hypertonic solutions (HOSM) on neurons to include effects on [Ca2+]i. We have looked for Ca(2+)-independent or -dependent neurosecretion evoked by these agents in parasympathetic postganglionic neurons dissociated from chick ciliary ganglia and maintained in tissue culture. The change in concentration of free Ca2+ in the micromolar range inside neurons ([Ca2+]i) was measured with indo-1 with the use of a Meridian ACAS 470 laser scanning microspectrophotometer. 3. Elevated concentration of extracellular KCl increased [Ca2+]i and the frequency of quantal events. Also, a twofold increase in osmotic pressure (HOSM) produced a similar increase in quantal release and a significant rise in [Ca2+]i; however, the Ca2+ appeared to come from intracellular stores. 4. In contrast, ETOH stimulated quantal neurosecretion without a measurable change in [Ca2+]i. It appears the alcohol exerts its influence on some stage in the process of exocytosis that is distal to or independent of the site of Ca2+ action. 5. The effects of high [KCl]o and osmotic pressure were occlusive. This is explained in part by the observation that hypertonicity reduced Ca2+ current, but an action on Ca2+ stores is also likely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of BK channels in rat chromaffin cells requires summation of Ca(2+) influx from multiple Ca(2+) channels

Large-conductance Ca(2+) and voltage-dependent K(+) channels (BK channels) in many tissues require high Ca(2+) concentrations for activation and therefore might be expected to be tightly coupled to Ca(2+) channels. However, in most cases, little is known about the relative organization of the BK channels and the Ca(2+) channels involved in their activation. We probed the nature of the organization of BK and Ca(2+) channels in rat chromaffin cells by manipulating Ca(2+) influx through Ca(2+) channels and by altering cellular Ca(2+) buffering using EGTA and bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid (BAPTA). The results were analyzed to determine the distance between Ca(2+) and BK channels that would be most consistent with the experimental data. Most BK channels are close enough to Ca(2+) channels to be resistant to the buffering action of millimolar of EGTA, but are far enough to be inhibited by BAPTA. Analysis of the EGTA/BAPTA results suggests that BK channels are at a distance of 50 to 160 nm from Ca(2+) channels. A model that assumes random distribution of Ca(2+) and BK channels fails to account for the observed [Ca(2+)](i) detected by BK channels, suggesting that a specific mechanism may exist to mediate the functional coupling between these channels. Importantly, the effects of EGTA and BAPTA cannot be explained by assuming a one-to-one coupling between Ca(2+) and BK channels. Rather, Ca(2+) influx through a number of Ca(2+) channels appears to act in concert to regulate the behavior of any individual BK channel. Thus differences in BK channel open probabilities may be explained by differences in the extent of Ca(2+) domain overlap at the sites of individual BK channels.     

Intracellular [Ca(2+)] transients in Ca(2+) wave in single rat ventricular myocyte

Ca(2+) release from the sarcoplasmic reticulum (SR) in heart muscle grades depending on Ca(2+) influx in the physiological twitch; Ca(2+( wave results from regenerative Ca(2+) release from the SR. To examine if the Ca(2+) release from the SR in the Ca(2+) wave takes a duration similar to the physiological one, a transient rise of intracellular [Ca(2+)] ([Ca(2+)](i) transient) was recorded during both a propagating Ca(2+) wave and an electrically evoked twitch with single rat ventricular myocytes, using a laser scanning confocal microscope. Care was taken to record the fluo-3 fluorescence from a segmental region with little lateral movement, especially during a propagating Ca(2+) wave. During a typical Ca(2+) wave, the time-to-peak (TP) and the half-width (HD) of the averaged [Ca(2+)](i) transient were 161 and 253 ms respectively, but they were 76 and 145 ms during an electrically evoked twitch. The difference in the duration between the two types of [Ca(2+)](i) transients could not be accounted for by modification of duration of [Ca(2+)](i) transient by possible asynchronous Ca(2+) release from the SR during a Ca(2+) wave, suggesting that the regenerative Ca2+) release from the SR in the Ca2+) wave occurs more slowly than the physiological one in rat ventricular myocytes.     

Somatic Ca(2+) dynamics in response to choline-mediated excitation in histaminergic tuberomammillary neurons

Histaminergic tuberomammillary (TM) neurons of the posterior hypothalamus have been implicated in cognition, alertness and sleep-wakefulness cycles. Spontaneous firing of TM neurons has been associated with histamine release and wakefulness. The expression of alpha7 nicotinic acetylcholine receptors (nAChRs) in TM neurons suggests a role for endogenous choline and for nicotinic drugs in the regulation of intracellular Ca(2+) metabolism, normal TM neuronal activity and histamine release. First, we established the link between TM neuronal spontaneous firing frequency and cytosolic free Ca(2+) concentration ([Ca(2+)](i)). A strong correlation was observed: an onset of spontaneous firing (3-4Hz) was accompanied by a 20-fold increase in [Ca(2+)](i) from 56+/-18 nM to 1.0+/-0.6 microM. The same range of firing frequencies has been observed in TM neurons in vivo and is associated with wakefulness. Secondly, choline-induced activation of alpha7 nAChRs did not elevate [Ca(2+)](i) directly, i.e. in the absence of high-threshold voltage-gated Ca(2+) channel (HVGCC) activation. Cd(2+) (200 microM) completely blocked all Ca(2+) signals, but inhibited only 37+/-16% of alpha7 nAChR-mediated currents. Thirdly, the responsiveness of [Ca(2+)](i) to choline-mediated excitation was inhibited by hyperpolarization and enhanced by depolarization, sensitizing [Ca(2+)](i) at membrane voltages associated with normal TM neuronal activity. These properties of [Ca(2+)](i) define the ability of TM neurons to translate cholinergic stimuli of identical strengths into different cytosolic Ca(2+) effects, providing the physiological substrate for state-specific modulation of incoming cholinergic information and would be expected to play a very important role in determining activity profiles of TM neurons exposed to elevated concentrations of cholinergic agents, such as choline and nicotine.     

Photolytic manipulation of Ca2+ and the time course of slow, Ca(2+)-activated K+ current in rat hippocampal neurones.

1. Experiments were performed on hippocampal CA1 pyramidal cells to investigate the time course of a slow, Ca(2+)-activated K+ current that follows a burst of action potentials. At a temperature of 27-30 degrees C, this current rises to a peak 200-400 ms following the cessation of Ca2+ entry before decaying to baseline in 4-8s. 2. Intracellular recordings were made using electrodes containing the photolabile calcium buffers nitr-5 or DM-nitrophen loaded appropriately with Ca2+. Under these conditions, photolysis of the compound using an ultraviolet flashlamp caused an instantanous increase in cytoplasmic Ca2+ throughout the cell. The response to flash photolysis was a membrane hyperpolarization with an onset limited by the membrane time constant. Multiple (up to twenty) flash responses could be generated. 3. The postspike slow after-hyperpolarization (AHP) and flash-induced hyperpolarizations showed a common sensitivity to the beta-adrenergic receptor agonist isoprenaline. 4. Following a burst of spikes, the current underlying an AHP in progress could be terminated or reduced by photolysis-induced production of calcium buffer from diazo-4 within the cell. This action was rapid (within the setting time of the flash artifact, i.e. < 10 ms) despite the fact that the manipulation occurred 400-500 ms following the end of Ca2+ entry. 5. Partial block of the slow AHP by buffer production was accompanied by an increase in the time to peak of the event. 6. The time to peak of the slow AHP could also be manipulated by experiments which altered the spatial distribution of Ca2+ entry, such as production of calcium spikes or dendritic depolarization by glutamate in the presence of tetrodotoxin. 7. The Ca(2+)-dependent K+ current responsible for the slow AHP responds immediately to increase or decreases in cytoplasmic Ca2+. It seems likely, therefore, that the slow AHP is controlled solely by changes in free Ca2+ and that the time course is governed by the redistribution of cytoplasmic Ca2+ following activity-induced entry through voltage- or receptor-operated channels.     

Characterization of Ca(2+) channels in rat subthalamic nucleus neurons

The subthalamic nucleus (STN) plays a key role in motor control. Although previous studies have suggested that Ca(2+) conductances may be involved in regulating the activity of STN neurons, Ca(2+) channels in this region have not yet been characterized. We have therefore investigated the subtypes and functional characteristics of Ca(2+) conductances in STN neurons, in both acutely isolated and slice preparations. Acutely isolated STN cells were identified by retrograde filling with the fluorescent dye, Fluoro-Gold. In acutely isolated STN neurons, Cd(2+)-sensitive, depolarization-activated Ba(2+) currents were observed in all cells studied. The current-voltage relationship and current kinetics were characteristic of high-voltage-activated Ca(2+) channels. The steady-state voltage-dependent activation curves and inactivation curves could both be fitted with a single Boltzmann function. Currents evoked with a prolonged pulse, however, inactivated with multiple time constants, suggesting either the presence of more than one Ca(2+) channel subtype or multiple inactivation processes with a single channel type in STN neurons. Experiments using organic Ca(2+) channel blockers revealed that on average, 21% of the current was nifedipine sensitive, 52% was sensitive to omega-conotoxin GVIA, 16% was blocked by a high concentration of omega-agatoxin IVA (200 nM), and the remainder of the current (9%) was resistant to the co-application of all blockers. These currents had similar voltage dependencies, but the nifedipine-sensitive current and the resistant current activated at slightly lower voltages. omega-Agatoxin IVA at 20 nM was ineffective in blocking the current. Together, the above results suggest that acutely isolated STN neurons have all subtypes of high-voltage-activated Ca(2+) channels except for P-type, but have no low-voltage-activated channels. Although acutely isolated neurons provide a good preparation for whole cell voltage-clamp study, dendritic processes are lost during dissociation. To gain information on Ca(2+) channels in dendrites, we thus studied Ca(2+) channels of STN neurons in a slice preparation, focusing on low-voltage-activated channels. In current-clamp recordings, a slow spike was always observed following termination of an injected hyperpolarizing current. The slow spike occurred at resting membrane potentials and was sensitive to micromolar concentrations of Ni(2+), suggesting that it is a low-threshold Ca(2+) spike. Together, our results suggest that STN neurons express low-voltage-activated Ca(2+) channels and several high-voltage-activated subtypes. Our results also suggest the possibility that the low-voltage-activated channels have a preferential distribution to the dendritic processes.     

Effect of extracellular [Ca(2+)] on Ca(2+) release from sarcoplasmic reticulum in rat ventricular myocytes

The effect of external [Ca(2+)] ([Ca(2+)](o)) on Ca(2+) release from the sarcoplasmic reticulum (SR) was examined with rested-state twitches in rat ventricular myocytes. The magnitude of transient rise of intracellular [Ca(2+)] ([Ca(2+)](i)) relative to the resting one, F/F(o), as measured with fluo-3, was 1.75+/-0.07 (mean+/-SEM, n=9) and 1.86+/-0.13 (n=9) at 0.3 and 1.8 mM [Ca(2+)](o), respectively; the difference was insignificant. The time from onset to peak and the rate of rise of the [Ca(2+)](i) transient were 0.107+/-0.017 s (n=9) and 18.8+/-3.38 F/F(o)/s, respectively, at 0.3 mM [Ca(2+)](o), they were 0.064+/-0.005 s (n=9) and 31.1+/-0.03 F/F(o)/s, respectively, at 1.8 mM [Ca(2+)](o). The difference in the corresponding values at the two [Ca(2+)](o) was significant (t-test, p<0.05). The half decay time of the [Ca(2+)](i) transient was 0.217+/-0.016 s (n=8) at 0.3 mM [Ca(2+)](o) and was similar to the value of 0.230+/-0.022 s (n=8) at 1.8 mM [Ca(2+)](o), indicating that the rate of decrease of [Ca(2+)](i) is independent of the [Ca(2+)](o). The duration of action potential was similar at 0.3 and 1.8 mM [Ca(2+)](o) as examined with papillary muscle. The results suggest that a lowering of [Ca(2+)](o), i.e., reducing the Ca(2+) influx, slows the rate of Ca(2+) release from the SR fully loaded with Ca(2+) with little effect on the total amount of the Ca(2+) release. An instantaneous relationship between the [Ca(2+)](i) and the myocyte shortening at 0.3 and 1.8 mM [Ca(2+)](o) suggested that the time course of unloaded contraction is related not only to the magnitude, but also to the rate of rise of [Ca(2+)](i).     

Ca(2+)-independent but voltage-dependent secretion in mammalian dorsal root ganglion neurons

We have investigated the Ca(2+) dependence of vesicular secretion from the soma of dorsal root ganglion (DRG) neurons, which secrete neuropeptides by exocytosis of dense-core vesicles. In patch-clamped somata of rat DRG neurons, we found a depolarization-induced membrane capacitance increase (DeltaC(m)) in the absence of extracellular Ca(2+) and in the presence of a Ca(2+) chelator (BAPTA) in the intracellular solution. Depletion of internal Ca(2+) stores by thapsigargin in the Ca(2+)-free bath also did not block the DeltaC(m), indicating that Ca(2+) release from internal Ca(2+) stores may not have been involved. Furthermore, the Ca(2+)-independent DeltaC(m) was blocked by whole-cell dialysis with tetanus toxin and was accompanied by pulsatile secretion of false transmitters, as detected by amperometric measurements. These results indicate the existence of Ca(2+)-independent but voltage-dependent vesicular secretion (CIVDS) in a mammalian sensory neuron.     

Effects of Ca2+ antagonists and antiepileptics on tetrodotoxin-sensitive Ca(2+)-conducting channels in isolated rat hippocampal CA1 neurons.

All Ca2+ antagonists blocked tetrodotoxin-sensitive Ca2+ current (TTX-ICa) more potently than Na+ current (INa). Phenytoin and MK-801, at concentrations which had no effect on INa, could block TTX-ICa concentration-dependently. Valproic acid and phenobarbital had no effect on both TTX-ICa and INa. In particular, flunarizine and phenytoin have more potent inhibitory effects on TTX-ICa than other test drugs. These results suggest that the abnormal excess-excitation of TTX-sensitive Ca(2+)-conducting channels may be one of the trigger factors generating epilepsy.     

Inter-bouton variability of synaptic strength correlates with heterogeneity of presynaptic Ca(2+) signals

The elevation of presynaptic calcium concentration is a crucial step in excitation-secretion coupling. However, the amplitudes of action-potential-induced presynaptic calcium transients can display high variability among different terminals. The aim of this study was to clarify whether, at individual boutons, synaptic strength correlates with the average amplitude of presynaptic calcium transients. Low-density collicular cultures were loaded with the calcium indicator Oregon Green bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) 1. Action potentials were blocked with tetrodotoxin. Presynaptic terminals were identified with FM4-64, a use-dependent vesicle marker. Presynaptic calcium influx was elicited by a focal electrical stimulation of single boutons. Whole cell patch-clamp and calcium imaging techniques were used to record GABAergic evoked inhibitory postsynaptic currents (eIPSCs) and presynaptic fluorescence changes in the stimulated terminal. To make the eIPSCs from different boutons comparable, they were normalized to the mean value of miniature IPSCs (mIPSCs) of the postsynaptic cell. Records from 47 boutons showed that eIPSCs varied between 0.5 and 3.0 and presynaptic calcium transients varied between 0.1 and 1.3. However, there was a strong correlation between the mean amplitudes of eIPSCs and presynaptic calcium responses. The eIPSC-[Ca(2+)](pre) relationship allows to use the amplitudes of presynaptic calcium transients as an indicator of release efficacy and, in a set of contacts made by one axon, to predict the relative impact of individual terminals.