Local uncaging of caged Ca(2+) reveals distribution of Ca(2+)-activated Cl(-) channels in pancreatic acinar cells.

In exocrine acinar cells, Ca(2+)-activated Cl(-) channels in the apical membrane are essential for fluid secretion, but it is unclear whether such channels are important for Cl(-) uptake at the base. Whole-cell current recording, combined with local uncaging of caged Ca(2+), was used to reveal the Cl(-) channel distribution in mouse pancreatic acinar cells, where approximately 90% of the current activated by Ca(2+) in response to acetylcholine was carried by Cl(-). When caged Ca(2+) in the cytosol was uncaged locally in the apical pole, the Cl(-) current was activated, whereas local Ca(2+) uncaging in the basal or lateral areas of the cell had no effect. Even when Ca(2+) was uncaged along the whole inner surface of the basolateral membrane, no Cl(-) current was elicited. There was little current deactivation at a high cytosolic Ca(2+) concentration ([Ca(2+)](c)), but at a low [Ca(2+)](c) there was clear voltage-dependent deactivation, which increased with hyperpolarization. Functional Ca(2+)-activated Cl(-) channels are expressed exclusively in the apical membrane and channel opening is strictly regulated by [Ca(2+)](c) and membrane potential. Ca(2+)-activated Cl(-) channels do not mediate Cl(-) uptake at the base, but acetylcholine-elicited local [Ca(2+)](c) spiking in the apical pole can regulate fluid secretion by controlling the opening of these channels in the apical membrane.

L-Type calcium channels in enterochromaffin cells from guinea pig and human duodenal crypts: an in situ study.

BACKGROUND & AIMS: This study has investigated stimulus-secretion coupling of enterochromaffin cells by studying the cellular location and function of voltage-gated Ca(2+) channels within small intestinal crypts. METHODS: Digital fluorescence imaging and electrochemical detection were used to measure intracellular Ca(2+) responses and serotonin (5-hydroxytryptamine [5-HT]) secretion in intact crypts isolated from guinea pig and human duodenum. RESULTS: In fluo-3-loaded crypts, electrical depolarization with high K(+) solution increased cytosolic free [Ca(2+)] only in single cells subsequently identified by immunocytochemistry as enterochromaffin cells. In guinea pig enterochromaffin cells, the L-type Ca(2+) channel agonist FPL 64176 (3 micromol/L) did not change resting intracellular [Ca(2+)] but potentiated the depolarization-evoked increase in [Ca(2+)] (298 +/- 72 nmol/L) by 19 +/- 3-fold. In the majority of human enterochromaffin cells, FPL 64176 alone increased resting [Ca(2+)] by 423 +/- 171 nmol/L. Secretion studies in guinea pig crypts showed that high K(+) and FPL 64176 caused a 12-fold increase in 5-HT release. Noradrenaline caused increases in both enterochromaffin cell [Ca(2+)] and 5-HT release. CONCLUSIONS: Using this approach, we have found that in duodenal crypts, enterochromaffin cells, but not other epithelial cells, contain L-type voltage-gated Ca(2+) channels involved in regulating 5-HT secretion. These data have implications for the pharmacological control of intestinal disorders involving enterochromaffin cell dysfunction.

Somatostatin peptides inhibit basolateral potassium channels in human colonic crypts.

Somatostatin is a powerful inhibitor of intestinal Cl(-) secretion. We used patch-clamp recording techniques to investigate the effects of somatostatin on low-conductance (23-pS) K(+) channels in the basolateral membrane of human colonic crypts, which are an important component of the Cl(-) secretory process. Somatostatin (2 microM) elicited a >80% decrease in "spontaneous" K(+) channel activity in cell-attached patches in nonstimulated crypts (50% inhibition = approximately 8 min), which was voltage-independent and was prevented by pretreating crypts for 18 h with pertussis toxin (200 ng/ml), implicating a G protein-dependent mechanism. In crypts stimulated with 100-200 microM dibutyryl cAMP, 2 microM somatostatin and its synthetic analog octreotide (2 microM) both produced similar degrees of K(+) channel inhibition to that seen in nonstimulated crypts, which was also present under low-Cl(-) (5 mM) conditions. In addition, 2 microM somatostatin abolished the increase in K(+) channel activity stimulated by 2 microM thapsigargin but had no effect on the thapsigargin-stimulated rise in intracellular Ca(2+). These results indicate that somatostatin peptides inhibit 23-pS basolateral K(+) channels in human colonic crypt cells via a G protein-dependent mechanism, which may result in loss of the channel's inherent Ca(2+) sensitivity.

Voltage inactivation of Ca2+ entry and secretion associated with N- and P/Q-type but not L-type Ca2+ channels of bovine chromaffin cells.

1. In this study we pose the question of why the bovine adrenal medullary chromaffin cell needs various subtypes (L, N, P, Q) of the neuronal high-voltage activated Ca2+ channels to control a given physiological function, i.e. the exocytotic release of catecholamines. One plausible hypothesis is that Ca2+ channel subtypes undergo different patterns of inactivation during cell depolarization. 2. The net Ca2+ uptake (measured using 45Ca2+) into hyperpolarized cells (bathed in a nominally Ca2+-free solution containing 1.2 mM K+) after application of a Ca2+ pulse (5 s exposure to 100 mM K+ and 2 mM Ca2+), amounted to 0.65 +/- 0.02 fmol cell-1; in depolarized cells (bathed in nominally Ca2+-free solution containing 100 mM K+) the net Ca2+ uptake was 0.16 +/- 0.01 fmol cell-1. 3. This was paralleled by a dramatic reduction of the increase in the cytosolic Ca2+ concentration, [Ca2+]i, caused by Ca2+ pulses applied to fura-2-loaded single cells, from 1181 +/- 104 nM in hyperpolarized cells to 115 +/- 9 nM in depolarized cells. 4. A similar decrease was observed when studying catecholamine release. Secretion was decreased when K+ concentration was increased from 1.2 to 100 mM; the Ca2+ pulse caused, when comparing the extreme conditions, the secretion of 807 +/- 35 nA of catecholamines in hyperpolarized cells and 220 +/- 19 nA in depolarized cells. 5. The inactivation by depolarization of Ca2+ entry and secretion occluded the blocking effects of combined omega-conotoxin GVIA (1 microM) and omega-agatoxin IVA (2 microM), thus suggesting that depolarization caused a selective inactivation of the N- and P/Q-type Ca2+ channels. 6. This was strengthened by two additional findings: (i) nifedipine (3 microM), an L-type Ca2+ channel blocker, suppressed the fraction of Ca2+ entry (24 %) and secretion (27 %) left unblocked by depolarization; (ii) FPL64176 (3 microM), an L-type Ca2+ channel 'activator', dramatically enhanced the entry of Ca2+ and the secretory response in depolarized cells. 7. In voltage-clamped cells, switching the holding potential from -80 to -40 mV promoted the loss of 80 % of the whole-cell inward Ca2+ channel current carried by 10 mM Ba2+ (IBa). The residual current was blocked by 80 % upon addition of 3 microM nifedipine and dramatically enhanced by 3 microM FPL64176. 8. Thus, it seems that the N- and P/Q-subtypes of calcium channels are more prone to inactivation at depolarizing voltages than the L-subtype. We propose that this different inactivation might occur physiologically during different patterns of action potential firing, triggered by endogenously released acetylcholine under various stressful conditions.

Fatty acids stimulate cholecystokinin secretion via an acyl chain length-specific, Ca2+-dependent mechanism in the enteroendocrine cell line STC-1.

1. The present study has investigated whether fatty acids directly influence peptide release from enteroendocrine cells using STC-1, a mouse intestinal endocrine tumour cell line, previously shown to release cholecystokinin (CCK) in response to other physiological stimuli. 2. Fatty acids elicited a chain length- and dose-dependent stimulation of CCK secretion. Dodecanoic acid (C12) was most effective, producing up to a 5-fold increase in CCK secretion. Fatty acids with less than ten carbon atoms did not increase secretion. The chain length dependence of these effects mimics closely fatty acid-induced CCK secretion previously observed in humans in vivo. 3. Esterification of C12 abolished CCK secretion, indicating a critical role for a free carboxyl group in eliciting secretion. In contrast, modification of the methyl terminus had no effect on C12-induced secretion. The non-metabolizable C12 analogue 2-bromododecanoic acid was equally effective. 4. C12 elicited a marked increase in intracellular calcium levels (200-300 nM) in STC-1 cells which was abolished by the L-type Ca2+ channel antagonist nicardipine. In contrast, C8 produced a smaller and more transient Ca2+ response. C12-induced CCK secretion was also blocked by nicardipine. 5. These data suggest that fatty acids can interact directly with enteroendocrine cells to stimulate CCK secretion via increases in intracellular calcium mediated primarily by L-type Ca2+ channels.

Capacitative Ca2+ entry into Xenopus oocytes is sensitive to omega-conotoxins GVIA, MVIIA and MVIIC.

We have studied capacitative Ca2+ entry into Xenopus oocytes by depleting intracellular Ca2+ stores with inositol 1,4,5-trisphosphate or thapsigargin. Capacitative Ca2+ entry was evoked by hyperpolarisation and monitored via the Ca(2+)-activated Cl- current. Hyperpolarisation-evoked currents increased with extracellular [Ca2+] in the range 0.9-5 mM, and were reversibly inhibited by extracellular Mg2+ (0.1-10 mM) by up to 60%. Currents were decreased by the voltage-gated Ca2+ channel antagonists omega-conotoxin GVIA, MVIIA and MVIIC (0.3-10 microM) and the inhibition of Ca2+ entry in individual oocytes by omega-conotoxins GVIA and MVIIA was highly heterogeneous, but not additive. Flunarizine (10 microM) and the imidazoles SK&F 96365 (10 microM), miconazole (40 microM) and econazole (40 microM) partly blocked Ca2+ entry. Ca2+ entry was unaffected by calciseptine (300 nM) or alpha-bungarotoxin (1 microM). The possibility that these compounds might inhibit the Ca(2+)-activated Cl- current rather than capacitative Ca2+ entry itself was examined by recording the Cl- current activated by the increase in [Ca2+]i activated by the flash photolysis of caged Ca2+. Eicosatetraynoic acid (2-10 microM) markedly inhibited, and La3+ (1 mM but not 100 microM) potentiated the increase in Ca(2+)-activated Cl- current. In contrast, omega-conotoxins and Mg2+ had no effect on the Ca(2+)-activated Cl- current itself. These findings support the hypothesis that capacitative Ca2+ entry into Xenopus oocytes occurs through channels with a pharmacology similar to that of neuronal non-L type voltage-gated Ca2+ channels.

Serotonergic effects of dotarizine in coronary artery and in oocytes expressing 5-HT2 receptors.

In strips of pig coronary arteries incubated in oxygenated Krebs-bicarbonate solution at 37 degrees C, dotarizine blocked the phasic contractions evoked by 5-HT (0.5 microM) or K+ depolarization (35 mM K+) with an IC50 of 0.22 and 3.7 microM, respectively. Flunarizine inhibited both types of contractions with IC50 values of 1.7 microM for 5-HT and 2.4 microM for K+ responses. In Xenopus oocytes injected with in vitro transcribed RNA encoding for 5-HT2A or 5-HT2C receptors, 5-HT (100 nM for 20 s) applied every 10 min caused, in both cases, a reproducible inward current through Ca2(+)-activated Cl- channels (ICl). Dotarizine inhibited the 5-HT2A response in a concentration-dependent manner, with an IC50 of 2.2 nM. In contrast, the 5-HT2C response was unaffected by 1 microM dotarizine and blocked around 62% by 10 microM of this drug. The ICl activated either by intracellular injection of inositol 1,4,5-trisphosphate (IP3) in oocytes or by direct photorelease of Ca2+ in DM-nitrophen-injected oocytes was unaffected by 10 microM dotarizine. It is concluded that dotarizine blocks 5-HT2A receptors with a high affinity; the compound is devoid of intracellular effects on any further steps of the transduction pathway (i.e., IP3 receptor). Contrary to flunarizine that blocks equally well the serotonergic and the K+ vascular responses, dotarizine exhibits 17-fold higher affinity for vascular 5-HT receptors. These findings might be relevant to an understanding of the mechanism involved in the use of dotarizine and flunarizine as prophylactic agents in migraine.

Different contributions of L- and Q-type Ca2+ channels to Ca2+ signals and secretion in chromaffin cell subtypes.

In this study, we investigated the contribution of different subtypes of voltage-dependent Ca2+ channels to changes in cytosolic free Ca2+ ([Ca2+]i) and secretion in noradrenergic and adrenergic bovine chromaffin cells. In single immunocytochemically identified chromaffin cells, [Ca2+]i increased transiently during high K+ depolarization. Furnidipine and BAY K 8644, L-type Ca2+ channel blocker and activator, respectively, affected the [Ca2+]i rise more in noradrenergic than in adrenergic cells. In contrast, the Q-type Ca2+ channel blocker omega-conotoxin MVIIC inhibited the [Ca2+]i rise more in adrenergic cells. omega-Agatoxin IVA (30 nM), which blocks P-type Ca2+ channels, had little effect on the [Ca2+]i signal. The N-type Ca2+ channel blocker omega-conotoxin GVIA similarly inhibited the [Ca2+]i rise in both cell types. The effects of furnidipine, BAY K 8644, and omega-conotoxin MVIIC on K+-evoked norepinephrine and epinephrine release paralleled those effects on [Ca2+]i signals. However, omega-conotoxin GVIA and 30 nM omega-agatoxin IVA did not affect the secretion of either amine. The data suggest that, in the bovine adrenal medulla, the release of epinephrine and norepinephrine are preferentially controlled by Q- and L-type Ca2+ channels, respectively. P- and N-type Ca2+ channels do not seem to control the secretion of either catecholamine.

Characteristics of two basolateral potassium channel populations in human colonic crypts.

The basolateral membrane of human colonic crypt cells contains Ca2+ and cAMP activated, Ba2+ blockable, low conductance (23 pS) K+ channels, which probably play an important part in intestinal Cl- secretion. This study has defined more clearly the basolateral K+ conductive properties of human colonic crypts using patch clamp recording techniques. High conductance (138 pS) K+ channels were seen in 25% of patches (one or two channels per patch), and significantly inhibited by the addition of 5 mM Ba2+, 1 mM quinidine or 20 mM tetraethylammonium chloride (TEA) to the cytosolic side of excised inside-out patches, whereas 1 mM diphenylamine-2-carboxylic acid (DPC) had no effect. In contrast, clusters of the 23 pS K+ channel (two to six channels per patch) were present in > 75% of patches, and channel activity was inhibited by quinidine and DPC, but not by TEA. Activity of the 138 pS K+ channel in inside-out patches was abolished almost completely by removal of bath Ca2+, but in contrast with its effect on the 23 pS K+ channel, addition of 0.1 mM carbachol had no effect on the 138 pS K+ channel in cell attached patches. It is concluded that human colonic crypt cells possess two discrete basolateral K+ channel populations, which can be distinguished by their responses to K+ channel blockers, and their different sensitivities to changes in intracellular Ca2+ concentration.

Comparison of aldosterone- and RU-28362-induced apical Na+ and K+ conductances in rat distal colon.

In mammalian distal colon, aldosterone induces electrogenic Na+ absorption and electrogenic K+ secretion, whereas the sole transport effect of specific glucocorticoid agonists is thought to be stimulation of electroneutral NaCl absorption. In this study, intracellular microelectrodes and Na(+)- and K(+)-channel blockers were used to compare the effects of aldosterone and RU-28362 (a specific glucocorticoid agonist) on apical Na+ and K+ conductances in surface cells and upper crypt cells in the most distal colonic segment from adrenalectomized rats. In control animals, surface cells and crypt cells were devoid of apical Na+ and K+ conductances. In aldosterone-treated animals (70 micrograms.100 g body wt-1.day-1 for 7 days), Na+ conductances were induced in 88% of surface cells but only 40% of crypt cells, and the distribution of K+ conductances was similar (82% of surface cells and 50% of crypt cells). The same dose of RU-28362 also induced Na+ conductances in 82% of surface cells and 50% of crypt cells, which tended to be smaller than those induced by aldosterone. RU-28362, in contrast to aldosterone, had no effect on apical K+ conductance in surface cells or crypt cells. Concurrent treatment with the mineralocorticoid antagonist RU-28318 (3.5 mg.100 g body wt-1.day-1 for 7 days) inhibited Na(+)-channel expression in aldosterone-treated animals but had no effect in RU-28362-treated animals. We conclude that in the most distal segment of rat colon, aldosterone acts via mineralocorticoid receptors to induce apical Na+ and K+ conductances, which are only fully expressed in the surface cell population.(ABSTRACT TRUNCATED AT 250 WORDS)

Potassium channels in colonic crypts.

A characteristic feature of intestinal epithelia is their ability to secrete chloride (Cl-), a process that occurs mainly in intestinal crypts and is the critical transport event in secretory diarrhoea. Increased potassium (K+) channel activity in the basolateral membrane has an important role in the Cl- secretory process by hyperpolarising the cell and maintaining a favourable electrochemical driving force for Cl- exit at the apical membrane. We have shown, using patch-clamp techniques, that the basolateral membrane of human colonic crypt cells contains low conductance K+ channels that are voltage and calcium (Ca2+) sensitive and blocked by barium (Ba2+). These K+ channels are regulated by cytosolic cyclic adenosine monophosphate (cAMP) and Ca2+, intracellular second messengers that also stimulate Cl- secretion. This population of human intestinal K+ channels may be a target for the pharmacological control of Cl- secretory diarrhoea.

Aldosterone-induced apical Na+ and K+ conductances are located predominantly in surface cells in rat distal colon.

Aldosterone is a major regulator of Na(+)-absorptive and K(+)-secretory processes in the distal segment of mammalian colon. In this study, the distribution of aldosterone-sensitive cell types in isolated rat distal colon was determined using site-directed intracellular microelectrodes, specific Na(+)- and K(+)-channel blockers, and aldosterone-receptor binding techniques. Electrophysiological data indicated that aldosterone induced parallel apical membrane Na+ and K+ conductances, mainly in surface cells and to a significantly lesser degree in crypt cells. Scatchard analyses of aldosterone-receptor binding in cytosolic fractions revealed the maximum number of specific binding sites in whole mucosal homogenate and in the upper one-third and lower two-thirds of isolated crypt units to be 74.9 +/- 2.0, 59.8 +/- 2.4, and 59.3 +/- 3.2 fmol/mg protein, respectively, indicating the presence of aldosterone receptors in the crypt cell population. We conclude that in rat distal colon aldosterone-induced Na+ and K+ conductances (and by inference, electrogenic Na(+)-absorptive and K(+)-secretory processes) are located predominantly in the surface cell population and to a lesser extent in crypt cells, which also contain aldosterone receptors. This spectrum of aldosterone-induced Na+ and K+ conductances may reflect varying stages of differentiation along the surface cell-crypt cell axis.

The effects of hypo- and hyperthyroidism on fibre composition and mitochondrial enzyme activities in rat skeletal muscle.

Hypo- and hyperthyroidism have been associated with changes in the activities of mitochondrial enzymes in homogenates of skeletal muscles, but it is unclear whether such changes were due to changes in single fibre enzyme activities or to previously documented changes in relative numbers of fibres. In this study the activities of the mitochondrial enzymes alpha-glycerol phosphate dehydrogenase (m-alpha GPDH) and succinate dehydrogenase (SDH) were measured in single fibres of the soleus and gastrocnemius muscles of the rat by cytochemical assays. In the soleus muscles of hypothyroid animals there was a decrease in the mean percentage (+/- S.D.) of type II fibres from 8.0 +/- 6.0 to 0.8 +/- 1.9% (P less than 0.05) and decreases in SDH activities in all fibre types (P less than 0.005). In the gastrocnemius muscles of these animals there were no changes in fibre composition but type IIB fibres had reduced (P less than 0.05) m-alpha GPDH activities. In the hyperthyroid animals, in which body weight had increased relative to the euthyroid animals, there were increases in the percentages of type IC and type II fibres in the soleus from 4.3 +/- 1.7 to 13.1 +/- 9.0% (P less than 0.05) and from 9.6 +/- 7.2 to 33.4 +/- 9.6% (P less than 0.005) respectively and an increase in the percentage of type IIA fibres in the gastrocnemius from 92.9 +/- 2.3 to 97.0 +/- 2.9% (P less than 0.05). However, there were no increases in single fibre mitochondrial enzyme activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Tri-iodothyronine increases intra-cellular calcium levels in single rat myocytes.

We have studied the effects of acute administration of triiodothyronine (T3) on cytosolic free calcium levels [Ca2+]i in single rat myocytes microinjected with aequorin. Ventricular myocytes were isolated by perfusing rat hearts with collagenase, and healthy, rod-shaped cells were injected to less than 1% of their volume with aequorin. The photons emitted from single cells were measured and a conversion to [Ca2+]i made on the basis of an in vitro calibration after the remaining aequorin had been discharged by cell lysis. Only cells that depolarized reversibly (showing elevated [Ca2+]i levels) when superfused with 80 mM KCl, and which gave a substantial signal on lysis with distilled water were used. The [Ca2+]i rose from a resting value of 150 +/- 56 nM (mean +/- SD, n = 14) by 127 +/- 47 nM on depolarization with 80 mM KCl. Application of T3 (1-100 nM) led to an increase (P less than 0.05) in [Ca2+]i (mean amplitude of 152 +/- 35 nM) before returning to baseline. The median duration of these events was 10 min (range = 1.4-34.4 min). The time to response was shorter when 100 nM T3 was applied (median and range; 6.8, 0-14 min) than when 1 nM T3 was used (16, 7.0-56.1 min) (P less than 0.05). To conclude, physiological concentrations of thyroid hormones caused rapid but transient stimulation of [Ca2+]i in single rat myocytes.

Mitochondrial alpha-glycerol phosphate dehydrogenase activity in IIA fibres of the rat lateral gastrocnemius muscle; the effect of Ca2+ and ATP.

Mitochondrial alpha-glycerol phosphate dehydrogenase is an important enzyme, but it is difficult to extract and purify. We have measured the activity of this enzyme in single type IIA skeletal muscle fibres under initial rate conditions by microdensitometry of the formazan reaction product. The Km (1.6 mM) for the substrate (L-alpha-glycerol phosphate) was lower than reported for the extracted enzyme. Further, at low substrate concentrations (3 mM), the enzyme was allosterically activated by free Ca2+ concentrations of 1 microM or greater, and half-maximal stimulation occurred at 0.3 microM free Ca2+. In the absence of Ca2+, there was negative cooperativity of substrate binding with a Hill constant of 0.57, but no cooperativity occurred in the presence of calcium. ATP (10 mM) inhibited enzyme activity in the presence of Ca2+ but not in its absence.

Rapid continuous monitoring of enzyme activity in tissue sections: experience with the M85A and Zeiss UMSP-30 systems.

We have previously described methods for the continuous monitoring of formazan deposition in tissue sections with a system based on the Vickers M85A microdensitometer. However, this instrument only allows the monitoring of a single field in each section. We have now developed a new system based on the Zeiss UMSP-30 microspectrophotometer. This machine is entirely computer controlled and by virtue of its fast-scanning stage allows the rapid (less than 0.5 s) sequential monitoring of multiple fields (up to 35) in each section. Thus a number of cell types may be studied simultaneously and work which used to take a full working day with the M85A system now can be performed in 45 min. As with the M85A the Zeiss system has full capability for data analysis (i.e. calculation of initial velocity rates, etc.). We have found that continuous monitoring of tissue sections by microdensitometry is a precise, sensitive and biochemically valid method of studying enzyme activity within the cellular matrix.