In vitro splicing analysis showed that availability of a cryptic splice site is not a determinant for alternative splicing patterns caused by +1G-->A mutations in introns of the dystrophin gene.

BACKGROUND: Splicing patterns are critical for assessing clinical phenotype of mutations in the dystrophin gene. However, it is still unclear how to predict alternative splicing pathways in such cases of splice-site mutation in the dystrophin gene. OBJECTIVE: To identify elements determining alternative splicing pathways in intron +1G-->A mutations of the dystrophin gene. RESULTS: We found that exon 25 is spliced out in the +1G-->A mutation in intron 25, resulting in mild Becker muscular dystrophy, and that a cryptic splice site within exon 45 was activated in severe Duchenne muscular dystrophy with a mutation of +1G-->A mutation in 45. Furthermore, in vitro splicing analysis using a pre-constructed expression vector showed that the mutant intron 25 produced one transcript that lacked exon 25. In contrast, the same splice-site mutation in intron 45 produced three splicing products. One product used the same cryptic donor splice site within exon 45 as the in vivo donor site and another product used a cryptic splice site within the vector sequence. Notably, the available cryptic splice site was not activated by the same G-->A mutation of intron 25. CONCLUSION: It was concluded that sequences inserted into the in vitro splicing assay minigene contain cis-elements that determine splicing pathways. By taking other +1G-->A mutations in the introns of the dystrophin gene reported in the literature into consideration, it seems that cryptic splice-site activation is seen only in strong exons. This finding will help to elucidate the molecular pathogenesis of dystrophinopathy and to predict efficiency of induction of exon skipping with antisense oligonucleotides for treatment of Duchenne muscular dystrophy.

Dystrophin nonsense mutations can generate alternative rescue transcripts in lymphocytes.

Secondary alterations in splicing have been reported to produce semi-functional mRNA from several nonsense mutations in the dystrophin gene. Disruptions of exonic splicing enhancers by single nucleotide changes are thought to underlie such alterations. The precise frequencies of such nonsense mutation-dependent splicing alterations, however, remain unknown. Here we analyzed the splicing patterns of dystrophin mRNA in lymphocytes from 38 patients with dystrophinopathies due to nonsense mutations in the dystrophin gene. In seven of the cases (18%), we observed partial skipping of the nonsense-encoding exon. Two of the seven cases, however, exhibited complex activation of a nonsense mutation-created splice site, which resulted in the generation of novel transcripts. Examination of cis-regulatory splicing elements through calculation of splicing probability scores and identification of potential splicing enhancer or silencer sequences failed to disclose a single cause for exon skipping. Remarkably, individual differences in splicing patterns were observed for cells from patients with identical nonsense mutations (C.5899C>T). Although five cases produced semi-functional dystrophin mRNAs, only one of these exhibited a mild clinical course. These results provide important insights about targets for exon skipping induced by candidate antisense oligonucleotides and for ribosomal read-through of nonsense mutations.

T-type Ca2+ channels mediate propagation of odor-induced Ca2+ transients in rat olfactory receptor neurons.

Propagation of odor-induced Ca(2+) transients from the cilia/knob to the soma in mammalian olfactory receptor neurons (ORNs) is thought to be mediated exclusively by high-voltage-activated Ca(2+) channels. However, using confocal Ca(2+) imaging and immunocytochemistry we identified functional T-type Ca(2+) channels in rat ORNs. Here we show that T-type Ca(2+) channels in ORNs also mediate propagation of odor-induced Ca(2+) transients from the knob to the soma. In the presence of the selective inhibitor of T-type Ca(2+) channels mibefradil (10-15 microM) or Ni(2+) (100 microM), odor- and forskolin/3-isobutyl-1-methyl-xanthine (IBMX)-induced Ca(2+) transients in the soma and dendrite were either strongly inhibited or abolished. The percentage of inhibition of the Ca(2+) transients in the knob, however, was 40-50% less than that in the soma. Ca(2+) transients induced by 30 mM K(+) were partially inhibited by mibefradil, but without a significant difference in the extent of inhibition between the knob and soma. Furthermore, an increase of as little as 2.5 mM in the extracellular K(+) concentration (7.5 mM K(+)) was found to induce Ca(2+) transients in ORNs, and such responses were completely inhibited by mibefradil or Ni(2+). Total replacement of extracellular Na(+) with N-methyl-d-glutamate inhibited none of the odor-, forskolin/IBMX- or 7.5 mM K(+)-induced Ca(2+) transients. Positive immunoreactivity to the Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 subunits of the T-type Ca(2+) channel was observed throughout the soma, dendrite and knob. These data suggest that involvement of T-type Ca(2+) channels in the propagation of odor-induced Ca(2+) transients in ORNs may contribute to signal transduction and odor sensitivity.

Detection of oestrous-related odour in bovine ( Bos taurus) saliva: bioassay of identified compounds.

The present study was designed to identify the volatile constituents across the oestrous cycle of bovine in order to detect oestrous-specific chemical signal. The bovine saliva was extracted with diethyl ether (1 : 1 ratio, v/v) and analysed by gas chromatography-linked mass spectrometry. Numerous compounds were identified during oestrous cycle of bovine saliva. Among these, the compounds, namely, trimethylamine, acetic acid, phenol 4-propyl, pentanoic acid and propionic acid were specific to oestrous stage. The behaviour assay revealed that the compound, trimethylamine, is involved in attracting the male animal. The result concludes that the trimethylamine is considered as a putative oestrous-specific salivary chemo-signal in the bovine.

A physiological role for protoporphyrin IX photodynamic action in the rat Harderian gland?

AIMS: The lipid-secreting exocrine Harderian gland contains a large amount of porphyrins (mainly protoporphyrin IX, PPIX) in the glandular cells, the physiological significance of which is rather poorly understood. METHODS: In the present study, the possibility of using Fura-2 to measure intracellular calcium ([Ca2+]c) changes in these cells was assessed. RESULTS: It was found that when Fura-2-loaded cells were excited by light at 340/380 nm, [Ca2+]c increased spontaneously, indicating a photodynamic action powered by light at 340/380 nm. In contrast, with the visible spectrum calcium probe Fluo-3 (lambda(ex) = 475 nm), carbachol at 10 microm induced [Ca2+]c increase; [Ca2+]c did not change without carbachol stimulation. Brief illumination with light at 340/380 nm induced a large [Ca2+]c increase in Fluo-3-loaded cells. Photodynamic stimulation of [Ca2+]c increase was confirmed with an exogenous photosensitizer sulphonated aluminium phthalocyanine (SALPC) and visible light (>580 nm). The wavelength-dependence of the [Ca2+]c increase correlates well with the excitation spectrum of the isolated Harderian glandular cells. CONCLUSION: These data suggest that PPIX present in rat Harderian glandular cells plays the role of a photosensitizer which upon activation by UVA and blue components of daylight and subsequent singlet oxygen generation, triggers [Ca2+]c increase and secretory response. The PPIX photodynamic action may also play a potential role in photic entrainment of the central circadian clock.

Mutation in the prp12+ gene encoding a homolog of SAP130/SF3b130 causes differential inhibition of pre-mRNA splicing and arrest of cell-cycle progression in Schizosaccharomyces pombe.

prp12-1 is one of the mutants defective in pre-mRNA splicing at a nonpermissive temperature in Schizosaccharomyces pombe. We found that the prp12+ gene encodes a protein highly homologous with a human splicing factor, SAP130/SF3b130, a subunit of a U2 snRNP-associated complex SF3b. Prp12p was shown to interact genetically with Prp10p that is a homolog of SAP155/SF3b155, another subunit in SF3b, suggesting that Prp12p is a functional homolog of human SAP130/SF3b130. Prp12p tagged with GFP is uniformly localized in the nuclear DNA region. In addition to pre-mRNA splicing defects, the prp12-1 mutant produced elongated cells, a typical phenotype of cell division cycle (cdc) mutants, suggesting a possible link between pre-mRNA splicing and cell-cycle progression. We examined kinetics of splicing defects in prp12-1 and several other prp mutants using northern blot hybridization and found that, among all the tested pre-mRNAs, only Tflld pre-mRNA with low splicing efficiency showed detectable splicing defects at the nonpermissive temperature in prp12-1. In addition, we found that other prp mutants with the cdc phenotype also showed differential splicing defects in tested pre-mRNAs at the nonpermissive temperature. On the other hand, prp mutants that do not exhibit the cdc phenotype showed a rapid and complete block of pre-mRNA splicing in all the tested pre-mRNAs at the nonpermissive temperature, indicating that prp mutants with weak splicing defects have a tendency to exhibit the cdc phenotype. These results suggest that the cdc phenotype in prp12-1 is caused by a selective reduction of spliced transcripts encoding a protein (or proteins) required for G2/M transition.

Fission yeast Prp4p kinase regulates pre-mRNA splicing by phosphorylating a non-SR-splicing factor.

We provide evidence that Prp4p kinase activity is required for pre-mRNA splicing in vivo and show that loss of activity impairs G1-S and G2-M progression in the cell cycle. Prp4p interacts genetically with the non-SR (serine/arginine) splicing factors Prp1p and Prp5p. Bacterially produced Prp1p is phosphorylated by Prp4p in vitro. Prp4p and Prp1p also interact in the yeast two-hybrid system. In vivo labelling studies using a strain with a mutant allele of the prp4 gene in the genetic background indicate a change in phosphorylation of the Prp1p protein. These results are consistent with the notion that Prp4p kinase is involved in the control of the formation of active spliceosomes, targeting non-SR splicing factors.

Photodynamic action inhibits compound 48/80-induced exocytosis in rat peritoneal mast cells.

Photostimulation of sulfonated aluminum phthalocyanine (SALPC)-loaded mast cells (20,000 lux, 2 min) itself caused neither exocytosis nor [Ca2+]i increase in isolated rat peritoneal mast cells. This result is incompatible with that reported in other cell types such as pancreatic acinar cells. Stimulation with 50 micrograms/ml compound 48/80, a direct G-protein activator, induced massive exocytosis which was easily detectable under conventional microscope. The fluorescent granules stained with sulforhodamine B were found to be numerous on the perimetry of mast cells, confirming occurrence of exocytosis. The stimulation also increased [Ca2+]i and cell volume before initiation of exocytosis. Pretreatment of the cells with photodynamic action with 5 microM SALPC inhibited the compound 48/80-induced exocytosis, but the [Ca2+]i increase and the increase of cell volume were unaffected. NaN3 at 0.5 mM could relieve the photodynamic action-induced inhibition of exocytosis. These results indicate that, unlikely to other secretory or contractile cells, photodynamic action with SALPC does not directly affect exocytotic machinery but modulates some functional proteins involved in signal transduction process which may be posterior to G-protein activation in mast cells. Singlet oxygen may be involved in the photodynamic action-induced modulation. A possible target protein can be a protein in the cell membrane which binds with a protein of a granular membrane during the course of exocytosis.

Photodynamic modulation of adrenergic receptors in the isolated rat hepatocytes.

In isolated rat hepatocytes, noradrenaline (NA) 50 nM induced intracellular calcium ([Ca(2+)](c)) increase as (i) oscillations with each down-stroke of the spike reaching baseline, (ii) phasic increase with gradual decay, and (iii) phasic increase transforming into oscillations. At 25 nM and 50 nM, NA predominantly induced oscillatory increases; at 100 nM and 1 microM, phasic increases were predominant. Photodynamic action (30 s) with photosensitizer sulphonated aluminium phthalocyanine (SALPC, 5 microM) induced [Ca(2+)](c) increase as (i) no change, (ii) a single spike, or (iii) phasic increase. [Ca(2+)](c) oscillations induced by NA 50 nM were obliterated by photodynamic action (30 s), but when NA 200 nM, which normally induced plateau increases, was added to the now quiescent cells, [Ca(2+)](c) oscillations reemerged. These data indicate that photodynamic action could efficiently desensitize adrenergic receptors in hepatocytes. Photodynamic action may do so by crosslinking neighboring receptors or neighboring transmembrane domains of the same receptor.

Exocytosis in the dissociated pancreatic acinar cells of the guinea pig directly visualized by VEC-DIC microscopy.

To elucidate the detailed process of exocytosis at the highest possible accuracy, we dissociated the pancreatic acinus of the guinea pig and observed zymogen granules under a video-enhanced contrast differential interference contrast (VEC-DIC) microscope. The preparation was thin enough to resolve each zymogen granule with the best clarity. When acinar cells were stimulated with ACh (20 microM), many zymogen granules near the lumen showed an abrupt light intensity change. For a period of 10 s immediately before exocytosis, zymogen granules neither shifted their position nor altered their shape within an accuracy of 38 nm. The time required for individual granules to change the light intensity (the releasing time) ranged from 0.15 to 0.70 s. After each response, the granule maintained its altered contrast for a few seconds until it was retrieved to a planar membrane. No compound exocytosis including granule-granule fusion was observed. We concluded that the exocytosis is not directly initiated by any supramolecular change but by a purely molecular event.

Mutations in the large subunit of U2AF disrupt pre-mRNA splicing, cell cycle progression and nuclear structure.

The prp2 gene of fission yeast has previously been shown to encode the large subunit of the splicing factor spU2AF. SpU2AF(59) is an evolutionarily conserved protein that has an arginine/serine-rich region and three RNA recognition motifs (RRMs). We have sequenced three temperature-sensitive alleles of prp2 and determined that the mutations result in single amino acid changes within one of the RRMs or between RRMs. All mutant alleles of prp2 have pre-mRNA splicing defects at the non-permissive temperature. Although the mutant strains are growth-arrested at 37 degrees C, they do not elongate like typical fission yeast cell cycle mutants. The DNA of the prp2(-) strains stains more intensely than a wild-type strain, suggesting that the chromatin may be condensed. Ultrastructural studies show differences in the mutant nuclei including a prominent distinction between the chromatin- and non-chromatin-enriched regions compared to the more homogenous wild-type nucleus. Two-hybrid assays indicate that some of the wild-type protein interactions are altered in the mutant strains. These results suggest that normal functioning of spU2AF(59) may be essential not only for pre-mRNA splicing but also for the maintenance of proper nuclear structure and normal cell cycle progression.

Effects of AIF4- and ATP on intracellular calcium dynamics of crypt epithelial cells in mouse small intestine.

Previous digital imaging analysis of intracellular calcium ion ([Ca2+]i) dynamics in the crypt of the small intestine showed little response by most columnar cells to cholinergic and adrenergic agonists. The objective of the present study was to demonstrate whether G-protein activators and other transmitters elicit [Ca2+]i changes in crypt cells. We used digital imaging to analyze spatiotemporal dynamics of [Ca2+]i in Fura-2/AM-loaded isolated crypts of mouse duodenum and ileum. A1F4- increased [Ca2+]i in crypt columnar cells. In many cases, we observed [Ca2+]i oscillations, which were synchronized throughout the entire crypt. The oscillations were blocked by octanol. ATP, but not adenosine, caused a [Ca2+]i increase in middle crypt-regions of the duodenum and upper regions of the ileum, and the [Ca2+]i wave propagated towards the crypt bottom. The ATP-induced [Ca2+]i increase was prevented by pretreatment with thapsigargin or suramin, but not by La3+ or an extracellular Ca(2+)-free environment. Neither dopamine, 5-hydroxytryptamine (5-HT), histamine, vasoactive intestinal peptide, substance P. cholera toxin, nor guanylin had significant effects. The [Ca2+]i dynamics of Paneth cells were independent of the AlF4(-)-induced synchronous oscillations of columnar cells and of the ATP-induced [Ca2+]i wave. In conclusion, crypt columnar cells have [Ca2+]i-dependent intracellular signaling mechanisms that are linked with G proteins, and by which the cells communicate with each other. ATP elicited [Ca2+]i mobilization from columnar cells via P2 receptors, although some regional differences were noted between the duodenum and ileum.

Acute oxidative stress modulates secretion and repetitive Ca2+ spiking in rat exocrine pancreas.

The effects of the oxidant tert-butylhydroperoxide (t-buOOH) on carbachol-stimulated pancreatic secretion in the vascularly perfused rat pancreas have been studied in parallel with [Ca2+]i signalling and amylase output in perifused rat pancreatic acinar cells. Perfusion of the pancreas with t-buOOH (0.1-1 mM) caused a rapid and irreversible inhibition of carbachol-stimulated (3x10-7 M) amylase and fluid secretion. Pre-perfusion of the pancreas with vitamin C and dithiothreitol or a cocktail of GSH and GSH-precursor amino acids provided only marginal protection against the deleterious effects of t-buOOH, even though GSH levels were elevated significantly. In perifused pancreatic acini, repetitive [Ca2+]i spikes evoked by carbachol (3x10-7 M) were sustained for 40 min. t-buOOH (1 mM) acutely increased the amplitude and duration of Ca2+ spikes, then attenuated Ca2+ spiking and subsequently caused a marked and sustained rise in [Ca2+]i. t-buOOH-induced alterations in carbachol-stimulated [Ca2+]i signalling and amylase release in perifused pancreatic acini were prevented by vitamin C. Although vitamin C restored impaired Ca2+ signalling and maintained amylase output in pancreatic acini, it seems likely that oxidative stress inhibits fluid secretion irreversibly in the intact pancreas, resulting in a loss of amylase output. Thus, perturbations in [Ca2+]i signalling may not fully explain the secretory block caused by oxidative stress in acute pancreatitis.

Contribution of Na+/Ca2+ exchanger to glucose-induced [Ca2+]i increase in rat pancreatic islets.

The present study was carried out to elucidate the role of the reverse mode of the Na+/Ca2+ exchanger in an increase in intracellular Ca2+ concentration ([Ca2+]i) induced by a stimulatory concentration of glucose in rat pancreatic islets. The effects of KB-R7943, a selective inhibitor of reverse Na+/Ca2+ exchanger, on Na+o removal-induced [Ca2+]i changes were examined by a microfluorimetric method using fura-2 in perifused preparations of isolated rat pancreatic islets. Na+o removal induced a rapid increase in [Ca2+]i under 100 or 5 mM K+ conditions, respectively. The increases in [Ca2+]i induced by Na+o removal were inhibited by KB-R7943. The net amount of the [Ca2+]i increases during Na+o removal (Delta[Ca2+]i), obtained by subtracting the KB-R7943-independent Delta[Ca2+]i in the presence of KB-R7943 from Delta[Ca2+]i in the absence of KB-R7943, was significantly increased when extracellular K+ was raised. Increasing the external glucose concentration from 3 to 20 mM caused a biphasic increase in [Ca2+]i, which exhibited a transient increase (first phase) followed by a sustained increase (second phase) in [Ca2+]i. KB-R7943 (10 microM) partially inhibited the second phase of the [Ca2+]i increase rather than the first phase. These results suggest that the increase in [Ca2+]i induced by Na+o removal may be enhanced when plasma membrane is depolarized, and consequently, Ca2+ influx through the reverse Na+/Ca2+ exchanger may partially contribute to the glucose-induced [Ca2+]i dynamics in rat pancreatic islet cells.

Effects of substance P on nicotine-induced intracellular Ca2+ dynamics in bovine adrenal chromaffin cells.

Substance P (SP) is colocalized with ACh in splanchnic nerves that innervate into adrenal medulla and the peptide has been shown to inhibit nicotinic agonists-induced catecholamine secretion. To elucidate the effects of SP on cytosolic Ca2+ dynamics, the present study was conducted using fura-2-loaded isolated bovine adrenal chromaffin cells. Stimulation of the cells with nicotine (10-100 microM) produced a rapid rise of cytosolic Ca2+ concentration ([Ca2+]i), the peak level of which increased in a dose-dependent manner, followed by a gradual decay. In the presence of 10 microM SP, the dose-response relationship of the peak levels shifted downward. Quantitative analyses implied that SP inhibits the nicotine-induced Ca2+ influx in a noncompetitive manner. Nicotinic acetylcholine receptor is composed of two major functional domains: an agonist-binding site and an ionophore or channel domain. Agonist binding activates ionophore/channel domain and causes mainly Na+ influx. This Na+ influx depolarizes the cell and activates voltage-dependent Ca2+ channels. Based on this fact, the present results indicate that SP dose not block nicotine binding sites but interferes with other sites of nicotinic receptor/channel molecule, most probably a channel domain. It was suggested that SP colocalized with ACh in splanchnic nerves functions as a physiological modulator of catecholamine secretion by non-competitively suppressing ACh-induced cytosolic Ca2+ dynamics in bovine adrenal chromaffin cells.

The fission yeast prp10(+) gene involved in pre-mRNA splicing encodes a homologue of highly conserved splicing factor, SAP155.

In the fission yeast Schizosaccharomyces pombe, 14 prp (pre-mRNAprocessing) mutants have been isolated to date. We cloned the prp10(+) gene by complementation of the temperature-sensitive growth of prp10. Five types of transcripts were found that were alternatively spliced with respect to two possible introns located in the 5'-terminal region. Three of them are probably functional and code for putative proteins of approximately 1200 amino acids. Proteins highly homologous to Prp10p are present in other organisms, one of which is a human spliceosome-associated protein SAP155, a subunit of the splicing factor complex SF3. The C-terminal two-thirds of Prp10p is highly conserved among species, and contains consensus repeats for the regulatory subunit A of protein phosphatase PP2A. A gene disruption experiment indicated that the prp10(+) gene is essential for viability in S.pombe. Prp10p tagged with GFP is predominantly localized in the nuclear DNA region. A series of deletions showed that the less conserved N-terminal region of approximately 300 amino acids in Prp10p is dispensable, although the corresponding region was thought to play important roles in the mammalian splicing system.

Muscarinic and nicotinic receptor-mediated Ca2+ dynamics in rat adrenal chromaffin cells during development.

To clarify when the cholinergic receptor-mediated secretion mechanism of developing adrenal chromaffin cells is expressed and becomes functional, morphological changes and intracellular calcium dynamics were studied by immunohistochemistry, electron microscopy, and Fura-2 digital image analysis. From embryonic day 14 to 16, adrenal medullary cells were immunoreactive to noradrenaline-synthesizing enzyme (dopamine beta-hydroxylase) but not to adrenaline-synthesizing enzyme (phenylethanolamine N-methyltransferase). These cells contained either no granules or just a few granules of high electron density. Exocytotic figures were rarely observed in cells of the control or in cells after carbamylcholine stimulation. Nerve fibers in the adrenal medulla contained either no clear vesicles or very few. Neither methacholine nor nicotine caused a change of intracellular Ca2+ in most chromaffin cells. From embryonic day 18 to 20, chromaffin cells were immunoreactive to both dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase and they contained relatively numerous secretory granules. Exocytotic figures were often seen in cells after carbamylcholine stimulation. The intra-adrenal nerve fibers contained numerous clear vesicles and a few dense-cored vesicles. Methacholine caused no rise of intracellular Ca2+, but nicotine induced a low to relatively high rise in many cells. From postnatal day 2 or 3 to postnatal week 1, numerous cells were immunoreactive to both dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase, whereas some cells were reactive to dopamine beta-hydroxylase alone. Chromaffin cells were divisible into noradrenaline cells and adrenaline cells based on the ultrastructural features of their granules. Methacholine induced a moderate rise of intracellular Ca2+ and nicotine caused a high rise in many chromaffin cells, whereas, in some chromaffin cells, methacholine induced no rise of intracellular Ca2+ and nicotine induced a high rise. These results suggest that morphological changes of the developing cells and the intra-adrenal nerve fibers are related to the expression of a cholinergic receptor-mediated secretion mechanism and that this mechanism via a nicotinic receptor-mediated Ca2+ signaling pathway precedes the muscarinic receptor-mediated one during development.

Effects of carbachol and catecholamines on ultrastructure and intracellular calcium-ion dynamics of acinar and myoepithelial cells of lacrimal glands.

The current study was carried out to investigate autonomic nervous control of secretory functions in the lacrimal gland. To distinguish the difference between the responses to cholinergic and adrenergic agonists in acinar and myoepithelial cells in the lacrimal gland of guinea pigs, the morphological and functional responses to the agonists were examined by electron microscopy and by digital-imaging analysis of the intracellular concentration of Ca2+ ([Ca2+]i) using fluorescent Ca2+-indicators (Fura-2/AM and Indo-1/AM). In the resting state, exocytosis was rare, and the [Ca2+]i in acinar and myoepithelial cells was low (less than 300 nM). Stimulation with carbachol (CCh) induced a rapid rise in [Ca2+]i reaching a peak level followed by gradual decay and an appearance of many exocytotic figures. Approximately 4-8 s after an initial increase of [Ca2+]i, myoepithelial cells commenced contraction. Noradrenaline or adrenaline induced an increase in [Ca2+]i and exocytosis in acinar cells, but caused no [Ca2+]i increase in myoepithelial cells. In a Ca2+-deficient environment, the responses to CCh in myoepithelial cells and those to noradrenaline in acinar cells were inhibited, whereas the responses to CCh in acinar cells remained unchanged. Isoproterenol caused no effect on [Ca2+]i dynamics, although it occasionally induced exocytosis. Different cellular signaling pathways may be involved in the responses in acinar and in myoepithelial cells to different agonists. Lacrimation mechanisms are redundant.

A novel aspect of photodynamic action: induction of recurrent spikes in cytosolic calcium concentration.

Effects of photodynamic action of gadolinium porphyrin-like macrocycle B (PLMGdB) on cytosolic Ca2+ concentration, [Ca2+]c, was investigated in isolated rat pancreatic acini. The PLMGdB alone or light alone (2 min) had no effect on [Ca2+]c. Cell-bound PLMGdB upon brief (0.5-2.0 min) light activation triggered recurrent spikes in [Ca2+]c. At lower PLMGdB concentration (100 nM) the spikes continued during the whole period of monitoring [Ca2+]c. At a higher concentration of 500 nM, the spikes continued for the first 40 min, followed by a gradual increase in basal [Ca2+]c upon which smaller spikes were superimposed. At 1 microM, the spikes continued for the first 20 min, after that spiking gradually degenerated into a plateau phase. In many aspects, photodynamically triggered spikes resembled spikes generated by physiological concentrations of cholecystokinin. The spikes triggered by photodynamic action were likely to be the result of the ignition of a physiological "chain reaction", because functional inositol-1,4,5-trisphosphate (IP3) receptors were required for spiking to occur. Two-aminoethoxydiphenylborate, an inhibitory modulator of IP3-triggered Ca2+ release from intracellular stores, effectively inhibited photodynamically generated spikes. Therefore photodynamic action appears to be able to permanently transfix a physiological process, leading to long-lasting pharmacological or therapeutic effects.