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Disruption of neuronal Ca(2+) homeostasis contributes to neurodegenerative diseases through mechanisms that are not fully understood. A polymorphism in CALHM1, a recently described ion channel that regulates intracellular Ca(2+) levels, is a possible risk factor for late-onset Alzheimer's disease. Since there are six potentially redundant CALHM family members in humans, the physiological and pathophysiological consequences of CALHM1 function in vivo remain unclear. The nematode Caenorhabditis elegans expresses a single CALHM1 homolog, CLHM-1. Here we find that CLHM-1 is expressed at the plasma membrane of sensory neurons and muscles. Like human CALHM1, C. elegans CLHM-1 is a Ca(2+)-permeable ion channel regulated by voltage and extracellular Ca(2+). Loss of clhm-1 in the body-wall muscles disrupts locomotory kinematics and biomechanics, demonstrating that CLHM-1 has a physiologically significant role in vivo. The motility defects observed in clhm-1 mutant animals can be rescued by muscle-specific expression of either C. elegans CLHM-1 or human CALHM1, suggesting that the function of these proteins is conserved in vivo. Overexpression of either C. elegans CLHM-1 or human CALHM1 in neurons is toxic, causing degeneration through a necrotic-like mechanism that is partially Ca(2+) dependent. Our data show that CLHM-1 is a functionally conserved ion channel that plays an important but potentially toxic role in excitable cell function.

Exaggerated intracellular Ca2+signaling is a robust proximal phenotype observed in cells expressing familial Alzheimer's disease (FAD)-causing mutant presenilins (PSs). The mechanisms that underlie this phenotype are controversial and theirin vivorelevance for AD pathogenesis is unknown. Here, we used a genetic approach to identify the mechanisms involved and to evaluate their role in the etiology of AD in two FAD mouse models. Genetic reduction of the type 1 inositol trisphosphate receptor (InsP3R1) by 50% normalized exaggerated Ca2+signaling observed in cortical and hippocampal neurons in both animal models. In PS1M146V knock-in mice, reduced InsP3R1 expression restored normal ryanodine receptor and cAMP response element-binding protein (CREB)-dependent gene expression and rescued aberrant hippocampal long-term potentiation (LTP). In 3xTg mice, reduced InsP3R1 expression profoundly attenuated amyloid β accumulation and tau hyperphosphorylation and rescued hippocampal LTP and memory deficits. These results indicate that exaggerated Ca2+signaling, which is associated with FAD PS, is mediated by InsP3R and contributes to disease pathogenesisin vivo. Targeting the InsP3signaling pathway could be considered a potential therapeutic strategy for patients harboring mutations in PS linked to AD.

Single-channel properties of the Xenopus inositol trisphosphate receptor (IP3R) ion channel were examined by patch clamp electrophysiology of the outer nuclear membrane of isolated oocyte nuclei. With 140 mM K+ as the charge carrier (cytoplasmic [IP3] = 10 microM, free [Ca2+] = 200 nM), the IP3R exhibited four and possibly five conductance states. The conductance of the most-frequently observed state M was 113 pS around 0 mV and approximately 300 pS at 60 mV. The channel was frequently observed with high open probability (mean P(o) = 0.4 at 20 mV). Dwell time distribution analysis revealed at least two kinetic states of M with time constants tau5 ms and approximately 20 ms; and at least three closed states with tau approximately 1 ms, approximately 10 ms, and1 s. Higher cytoplasmic potential increased the relative frequency and tau of the longest closed state. A novel "flicker" kinetic mode was observed, in which the channel alternated rapidly between two new conductance states: F1 and F2. The relative occupation probability of the flicker states exhibited voltage dependence described by a Boltzmann distribution corresponding to 1.33 electron charges moving across the entire electric field during F1 to F2 transitions. Channel run-down or inactivation (tau approximately 30 s) was consistently observed in the continuous presence of IP3 and the absence of change in [Ca2+]. Some (approximately 10%) channel disappearances could be reversed by an increase in voltage before irreversible inactivation. A model for voltage-dependent channel gating is proposed in which one mechanism controls channel opening in both the normal and flicker modes, whereas a separate independent mechanism generates flicker activity and voltage-reversible inactivation. Mapping of functional channels indicates that the IP3R tends to aggregate into microscopic (1 microm) as well as macroscopic (approximately 10 microm) clusters. Ca2+-independent inactivation of IP3R and channel clustering may contribute to complex [Ca2+] signals in cells.

Inhibition of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases by thapsigargin elicits [Ca2+]i oscillations in rat salivary gland (parotid) acinar cells which are similar to those activated by agonists but are nevertheless independent of inositol 1,4,5-trisphosphate (IP3) or IP3-sensitive Ca2+ stores (Foskett, J. K., Roifman, C. M., and Wong, D. (1991) J. Biol. Chem. 266, 2778-2782). Neither bafilomycin alone or together with monensin or chloroquine inhibited thapsigargin-induced [Ca2+]i oscillations, ruling out the involvement of vacuolar-type proton pumps or organellar acidity in the mechanisms underlying them. Acute inhibition of plasma membrane Ca(2+)-ATPase by 1 mM La3+ inhibited the decline of [Ca2+]i during the falling phase of the oscillation. Acute inhibition of plasma membrane Ca2+ influx by removal of extracellular Ca2+, membrane depolarization, or inorganic channel blockers immediately abolished oscillations, even when applied during the [Ca2+]i rising phase of the cycle. Ca2+ influx rate oscillated during [Ca2+]i oscillations, varying 1.5-13-fold during a cycle. Modification of the rate of Ca2+ influx, by titrating the extent of depletion of IP3-sensitive stores or manipulating extracellular [Ca2+], indicated that oscillations depended on a high rate of Ca2+ influx. In thapsigargin- or carbachol-treated cells which did not exhibit a sustained [Ca2+]i rise or [Ca2+]i oscillations, inhibition of Ca2+ influx activated plasma membrane Ca2+ permeability. Thus, agonist- and thapsigargin-induced [Ca2+]i oscillations in parotid acinar cells appear to be generated by plasma membrane-based mechanisms which involve periodic inactivation by [Ca2+]i of the Ca2+ release-activated Ca2+ influx pathway.

Patch clamp of the outer nuclear membrane of isolated Xenopus oocyte nucleus was used to measure the single-channel properties of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R). The observed channel was activated by IP3, inhibited by heparin, and Ca(2+)-selective, with ion permeabilities PCa:PK:PCl = 8:1:0.05. In symmetric KCl buffer, the channel was ohmic (113 picosiemens in 140 mM KCl) at low channel currents but rectified at higher positive currents. The nuclear IP3R exhibited three conductance substates: a main substate occurring approximately 90% of channel open time, a double substate with twice the main substate conductance and a third substate with half the main substate conductance, which was observed rarely. Channel open probability fluctuated over time and among nuclei. Mean open channel durations of the main and double substates were approximately 5 and 1 ms, respectively. Many channels exhibited periods of closure lasting seconds, and most inactivated permanently within 5 min of IP3 stimulation. These results provide the first characterization of the single-channel properties of the IP3R in its native membrane environment and demonstrate that patch clamp electrophysiology of intact nuclei can be used to directly record currents through the IP3R.

Cholecystokinin (CCK) and carbachol raise intracellular Ca2+ concentration ([Ca2+]i) in pancreatic acinar cells by elevating inositol 1,4,5-trisphosphate (IP3). CCK analogues JMV-180 and OPE stimulate fully efficacious enzyme secretion and [Ca2+]i oscillations but release Ca2+ from intracellular stores by apparently IP3-independent mechanisms in permeabilized acinar cells. In the present study, we investigated whether OPE mobilizes Ca2+ from IP3-sensitive Ca2+ stores and whether IP3 mediates such responses in single intact cells. OPE and JMV-180 similarly elevated IP3 to low levels compared with those elicited by 10 nM CCK. Depletion of IP3-sensitive stores by elevation of intracellular IP3 using carbachol, microinjection of a nonmetabolizable IP3 analogue, or exposure to thapsigargin, in the absence of extracellular Ca2+, depleted the same Ca2+ stores that were sensitive to OPE. In converse experiments, OPE depleted carbachol- or thapsigargin-sensitive stores, indicating that carbachol-, thapsigargin-, IP3-, and OPE-sensitive Ca2+ stores overlap completely and that stores mobilized by OPE are IP3 sensitive. To determine whether IP3 mediates responses to OPE, cells were microinjected with low-molecular-weight heparin, a competitive inhibited the rise of [Ca2+]i in response to carbachol, OPE, or JMV-180, whereas de-N-sulfated heparin, an inactive heparin, was without effect. These results indicate that CCK analogues release Ca2+ from IP3-sensitive Ca2+ stores by mechanisms involving the IP3 receptor.

Pancreatic acinar cells, a model cell type for the study of exocytosis in non-excitable cells, were used here to test the hypothesis that molecular mechanisms regulating exocytosis from neuronal and neuroendocrine cells may also be utilized in exocrine cells. Using specific antisera raised against vesicle-associated membrane protein (VAMP) isoforms 1 and 2, we have identified VAMP-2, but not VAMP-1, immunoreactive proteins on rat pancreatic acinar cell secretory (zymogen) granules. This 18-kDa protein co-migrated with rat brain synaptic vesicle VAMP-2. Tetanus toxin (TeTx) light chain caused complete cleavage of this protein, which was prevented by the addition of EDTA. This toxin also inhibited in a dose-dependent manner Ca(2+)-stimulated enzyme secretion by up to approximately 30% in streptolysin O-permeabilized acini. This effect of TeTx on secretion was prevented by the zinc endopeptidase inhibitor captopril or by boiling the toxin. Incomplete inhibition of secretion by TeTx may suggest that TeTx-insensitive or VAMP-2-independent mechanisms also regulate exocytosis. In support, TeTx light chain was without effect on Rab3AL (effector domain peptide of Rab3)-mediated potentiation of Ca(2+)-stimulated secretion. These results indicate that a TeTx-sensitive VAMP-2-like protein on zymogen granules participates in Ca(2+)-regulated pancreatic enzyme secretion and that undefined Rab3AL-activated mechanisms may act independently to regulate exocytosis.

Na+/H+ exchange (antiport) is a major pathway for the regulation of intracellular pH. Antiport activity is stimulated when suspended cells adhere to the substratum. In this report, immunofluorescence was used to study the subcellular localization of the ubiquitous NHE-1 isoform of the antiport. NHE-1 was not distributed homogeneously on the surface of the cells. Instead, antiports were found to accumulate along the border of lamellipodia and near the edge of finer processes. Dual immunofluorescence experiments demonstrated that vinculin, talin and F-actin are concentrated at sites of NHE-1 accumulation. A mutated construct of NHE-1 lacking residues 566-635 of the cytosolic domain also accumulated near marginal lamellae. In contrast, the focal distribution observed in adherent cells was not detectable in cells grown in suspension. Fluorescence ratio imaging was used to define the functional consequences of focal accumulation of NHE-1. In the steady state, the pH was virtually identical throughout the cytosol. Moreover, no pH gradients were found to develop when cells recovered from an acid load by activation of Na+/H+ exchange. This is probably because of the presence of high concentrations of mobile buffers in the cytosol. The focal accumulation of antiporters near the cell margins may be involved in stimulation by adherence and/or generation of local osmotic gradients.

The Cl− conductance of a mouse fibroblast cell line (LTK− cells) that was stably transfected with the human CFTR (cystic fibrosis transmembrane conductance regulator) complementary DNA was studied. Single Cl− channel activity was observed only after treatment of the cells with forskolin, the single-channel conductance being 6.2 ± 0.2 pS with a linear current–voltage relationship. In CFTR+ cells, the whole-cell current at +90 mV increased from 7.3 ± 2.7 pA/pF (n = 12) to 46.1 ± 11.2 pA/pF (n = 5) after addition of dibutyryl-cyclic AMP (10−4 M) to the bath. Increasing the intracellular Cl− concentration to 150 mM activated linear Cl− currents in the absence of cyclic AMP in CFTR+ (n = 42) but not in CFTR− cells (n = 4). Similar Cl− current was also activated by high intracellular I− concentration. These results indicate that the CFTR-induced Cl− conductance in LTK− cells can be activated by either cyclic AMP or high intracellular halide concentrations.Key words: cystic fibrosis transmembrane conductance regulator (CFTR), chloride channel, cyclic AMP, whole-cell patch clamp, single-channel patch clamp.

We describe a simple and rapid technique for assaying both constitutive and regulated plasma membrane Cl- conductances. The method uses right-angle light scattering to measure the rate of swelling of cells in suspension, in which the anion conductance is rate limiting for swelling, due to introduction of high plasma membrane cation conductance using gramicidin. The technique was verified using Chinese hamster ovary cells and mouse L cells, both stably transfected with the cystic fibrosis transmembrane conductance regulator (CFTR), to confer a specific cAMP-activated Cl- conductance not normally present in these cell types. In agreement with results obtained using other methods for assaying Cl- permeability in these cells, forskolin stimulated a significant increase in plasma membrane Cl- conductance in CFTR-expressing cells, as indicated by an increase in light scattering. That the enhanced light scattering by the cells was the result of cell swelling due to NaCl influx was shown by ion substitution experiments, in which no forskolin-induced increase in light scatter occurred in N-methyl-D-glucamine Cl- or Na+ gluconate medium. Enhanced light scattering was also observed in both CFTR-expressing and control cells stimulated with the Ca2+ ionophore, ionomycin. Extracellular anion substitution, to exploit the inwardly directed halide gradient utilized in this protocol, enabled determination of the anion selectivities of both the cAMP- and Ca(2+)-activated Cl- channels. Thus this technique provides a simple optical method for rapidly assaying not only constitutive and regulated Cl- conductance pathways but also their anion selectivities.

Previous studies have demonstrated in single rat parotid acinar cells that the microsomal Ca(2+)-ATPase inhibitor thapsigargin mobilizes Ca2+ specifically from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store, activates plasma membrane Ca2+ permeability, and induces intracellular Ca2+ concentration ([Ca2+]i) oscillations that are quite similar to those activated by carbachol. Nevertheless, the IP3-sensitive Ca2+ store remains continuously depleted during thapsigargin-induced oscillations, indicating that this pool is not involved in the oscillation mechanism. To determine the specificity of thapsigargin's effects, in the present study we have examined the effects on [Ca2+]i in single rat parotid acinar cells of two other microsomal Ca(2+)-ATPase inhibitors, cyclopiazonic acid (CPA) and 2,5-di-tert-butyl-1,4-benzohydroquinone (BHQ), and compared them with the effects of thapsigargin in the same cells. Our results demonstrate that thapsigargin, CPA, and BHQ all similarly deplete the IP3-sensitive Ca2+ store specifically, activate plasma membrane Ca2+ influx, and induce [Ca2+]i oscillations, strongly suggesting that these agents have a specific inhibitory action on microsomal Ca(2+)-ATPase activity. BHQ, in addition, inhibits plasma membrane Ca2+ influx. The data lend strong support to a model in which the state of Ca2+ filling of the IP3-sensitive store regulates plasma membrane Ca2+ influx. These results suggest either that a Ca2+ pump is involved which is insensitive to structurally dissimilar inhibitors or that a Ca2+ pump is not involved in refilling of the Ca2+ pool involved in [Ca2+]i oscillations in these cells.

The microsomal Ca-ATPase inhibitor thapsigargin induces in rat salivary acinar cells [Ca2+]i oscillations which, though similar to those activated by agonists, are independent of inositol phosphates or inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ stores (Foskett, J. K., Roifman, C., and Wong, D. (1991) J. Biol. Chem. 266, 2778-2782). To examine whether the oscillation mechanism resides in another, thapsigargin- and IP3-insensitive intracellular store, we examined the effects of caffeine and ryanodine, known modulators of Ca2+ release from sarcoplasmic reticulum in excitable cells. Oscillations were induced by caffeine (1-20 mM) in nonoscillating thapsigargin-treated acinar cells, which required the continued presence of caffeine, whereas caffeine was without effect or reduced oscillation amplitude in oscillating cells. Ryanodine (10-50 microM) inhibited oscillations in most of the cells. These results suggest that Ca2+ oscillations in parotid acinar cells are driven by periodic Ca2+ release from an IP3-insensitive Ca2+ store with properties similar to sarcoplasmic reticulum of excitable cells.

The tumor promoter thapsigargin releases Ca2+ from intracellular stores by specific inhibition of microsomal Ca-ATPase activity without inositol phosphate formation. Recent studies of the actions of thapsigargin support the concept that the level of Ca2+ within the inositol (1,4,5)-trisphosphate (IP3)-sensitive intracellular pool regulates the Ca2+ permeability of the plasma membrane. We examined the effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) in single rat parotid cells using digital fluorescence microscopy. In the absence of extracellular Ca2+ (Ca2+o), thapsigargin transiently increased [Ca2+]i. Following the thapsigargin-induced [Ca2+]i transient, carbachol in the continued absence of Ca2+o was unable to raise [Ca2+]i, indicating that thapsigargin mobilizes Ca2+ from the IP3-sensitive store. In the converse experiment, carbachol prevented a rise of [Ca2+]i by thapsigargin, suggesting that the IP3- and thapsigargin-sensitive Ca2+ pools are the same. Depletion of Ca2+ from the IP3-sensitive pool by thapsigargin enhanced plasma membrane Ca2+ permeability. Thapsigargin triggered sustained Ca2+ oscillations in Ca2(+)-containing medium which are highly reminiscent of agonist-induced oscillations in these cells. Carbachol addition rapidly raised IP3 levels during oscillations triggered by thapsigargin but did not elevate [Ca2+]i, indicating that the IP3-sensitive pool remains continuously depleted during [Ca2+]i fluctuations. The results from this study rule out the involvement of the IP3-sensitive pool in the mechanisms involved in thapsigargin-induced (and by analogy, agonist-induced) oscillations in parotid cells.

Mammalian cells, including leukocytes, tend to gain volume passively be­ cause of the presence of intracellular charged macromolecules, which are impermeant. This tendency of the cells to swell is counteracted by the active extrusion of monovalent ions, which results in the maintenance of cellular volume. In fact, many cell types preserve an approximately constant cell size in the face of variations in the osmolarity of the medium. The precise coupling between the inward ion leak and the active outward pumping under both isotonic and anisotonic conditions is unlikely to be fortuitous. Instead, the preservation of near-normal size under such a variety of conditions suggests that the cells are able to sense thei.r volume and to compensate for departure from an optimal, physiologic state. This implies the existence of volume­ sensitive ion transport pathways that mediate the net loss of solutes when enlarged cells are required to shrink and vice versa. Under physiologic conditions, such transport pathways are likely activated by minute departures from the normal volume. Though subtle and effective, such responses are obviously difficult to study. Instead, several groups have resorted to a more convenient experimental paradigm to investigate the regulation of cellular volume. In this approach, substantial volume changes are imposed by abrupt­ ly varying the tonicity of the medium, in the expectation that the responses

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP3R) is an endoplasmic reticulum-localized Ca2+ -release channel that controls complex cytoplasmic Ca(2+) signaling in many cell types. At least three InsP3Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP3R (r-InsP3R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP3R-3 exhibited a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP3R-3 gating was inhibited by high [Ca2+]i with features similar to those of the endogenous Xenopus type 1 Ins3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of approximately 3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 microM under saturating (10 microM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3]100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP(3) concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of approximately 4. InsP(3) activated the type 3 channel by tuning the efficacy of Ca2+ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP3R-3 channel was uniquely distinguished from the X-InsP3R-1 channel by its enhanced Ca2+ sensitivity of activation (half-maximal activating [Ca2+]i of 77 nM instead of 190 nM) and lack of cooperativity between Ca2+ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP3R-3 with high gain InsP3-induced Ca2+ release and low gain Ca2+ -induced Ca2+ release properties complementary to those of InsP3R-1. Thus, distinct Ca2+ signals may be conferred by complementary Ca2+ activation properties of different InsP3R isoforms.

To find more efficient vectors for the transfer of CFTR cDNA, lactosylated polylysine was explored for transfer into airway epithelial cells in primary culture. The efficacy and high efficiency of transfection were shown by several criteria: expression of both mRNA and protein for CFTR and the functional correction of the Cl- channel activity. Using specific combinations of agents to enhance the transfection, an efficiency of 90% was obtained as detected by in situ hybridization with digoxigenin-labeled probes generated against exon 14 of CFTR. The highest efficiency was observed by adding E5CA peptide (10 microg) and 5% glycerol to the transfection mixture. The degree of transfection could be controlled by the enhancing agents, thus modulating the efficiency of transfection. The highest level of transfection efficiency is equivalent to that reported for viral vectors. None of the agents or their combinations in the concentrations used were cytotoxic to the primary cells. Antibody pAb3145 was used to detect the expression of the CFTR protein in the cells. When an N-terminal GFP-CFTR fusion gene was used to transfect the CF cells a functional correction of the CFTR Cl- channel was detected by patch-clamp electrophysiology. The high efficiency of CFTR gene transfer with lactosylated polylysine leads to the conclusion that lactosylated polylysine is a promising vector to transfer the CFTR gene into human airway cells in culture.

Cholinergic agonists stimulate isotonic fluid secretion in the parotid gland. This process is driven by the apical exit of Cl-, which enters the cells partly via Cl-/HCO-3 exchange across the basolateral membrane. Acidification of the cytosol by the extrusion of HCO-3 is prevented by the concomitant activation of the Na+/H+ exchanger (NHE), which is directly activated by cholinergic stimulation. Multiple isoforms of the NHE have been described in mammalian cells, but the particular isoform(s) present in salivary glands and their mechanism of activation have not been defined. Reverse transcriptase-polymerase chain reaction with isoform-specific primers was used to establish that NHE-1 and NHE-2, but not NHE-3 or NHE-4, are expressed in parotid glands. The presence of NHE-1 was confirmed by immunoblotting and immunofluorescence, which additionally demonstrated that this isoform is abundant in the basolateral membrane of acinar cells. The predominant role of NHE-1 in carbachol-induced Na+/H+ exchange was established pharmacologically using HOE694, an inhibitor with differential potency toward the individual isoforms. Because muscarinic agonists induce stimulation of protein kinases in acinar cells, we assessed the role of phosphorylation in the activation of the antiport. Immunoprecipitation experiments revealed that, although NHE-1 was phosphorylated in the resting state, no further phosphorylation occurred upon treatment with carbachol. Similar phosphopeptide patterns were observed in control and carbachol-treated samples. Together, these findings indicate that NHE-1, the predominant isoform of the antiporter in the basolateral membrane of acinar cells, is activated during muscarinic stimulation by a phosphorylation-independent event. Other processes, such as association of Ca2+-calmodulin complexes to the cytosolic domain of the antiporter, may be responsible for the activation of Na+/H+ exchange.

Inhibition of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases by thapsigargin elicits [Ca2+]i oscillations in rat salivary gland (parotid) acinar cells which are similar to those activated by agonists but are nevertheless independent of inositol 1,4,5-trisphosphate (IP3) or IP3-sensitive Ca2+ stores (Foskett, J. K., Roifman, C. M., and Wong, D. (1991) J. Biol. Chem. 266, 2778-2782). Neither bafilomycin alone or together with monensin or chloroquine inhibited thapsigargin-induced [Ca2+]i oscillations, ruling out the involvement of vacuolar-type proton pumps or organellar acidity in the mechanisms underlying them. Acute inhibition of plasma membrane Ca(2+)-ATPase by 1 mM La3+ inhibited the decline of [Ca2+]i during the falling phase of the oscillation. Acute inhibition of plasma membrane Ca2+ influx by removal of extracellular Ca2+, membrane depolarization, or inorganic channel blockers immediately abolished oscillations, even when applied during the [Ca2+]i rising phase of the cycle. Ca2+ influx rate oscillated during [Ca2+]i oscillations, varying 1.5-13-fold during a cycle. Modification of the rate of Ca2+ influx, by titrating the extent of depletion of IP3-sensitive stores or manipulating extracellular [Ca2+], indicated that oscillations depended on a high rate of Ca2+ influx. In thapsigargin- or carbachol-treated cells which did not exhibit a sustained [Ca2+]i rise or [Ca2+]i oscillations, inhibition of Ca2+ influx activated plasma membrane Ca2+ permeability. Thus, agonist- and thapsigargin-induced [Ca2+]i oscillations in parotid acinar cells appear to be generated by plasma membrane-based mechanisms which involve periodic inactivation by [Ca2+]i of the Ca2+ release-activated Ca2+ influx pathway.

Patch clamp of the outer nuclear membrane of isolated Xenopus oocyte nucleus was used to measure the single-channel properties of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R). The observed channel was activated by IP3, inhibited by heparin, and Ca(2+)-selective, with ion permeabilities PCa:PK:PCl = 8:1:0.05. In symmetric KCl buffer, the channel was ohmic (113 picosiemens in 140 mM KCl) at low channel currents but rectified at higher positive currents. The nuclear IP3R exhibited three conductance substates: a main substate occurring approximately 90% of channel open time, a double substate with twice the main substate conductance and a third substate with half the main substate conductance, which was observed rarely. Channel open probability fluctuated over time and among nuclei. Mean open channel durations of the main and double substates were approximately 5 and 1 ms, respectively. Many channels exhibited periods of closure lasting seconds, and most inactivated permanently within 5 min of IP3 stimulation. These results provide the first characterization of the single-channel properties of the IP3R in its native membrane environment and demonstrate that patch clamp electrophysiology of intact nuclei can be used to directly record currents through the IP3R.

Fluid secretion by epithelial cells can be modulated by agents that activate Cl- channels in the apical membrane. To sustain secretion, Cl- influx across the basolateral membrane must also be accelerated. To examine the cellular mechanisms that couple Cl- efflux across the apical membrane to Na(+)-coupled Cl- entry across the basolateral membrane, we employed optical imaging techniques, utilizing single rat salivary acinar cells. Na+ influx was negligible in resting cells but was rapidly increased by carbachol due to activation of a Na(+)-H+ exchanger, a Na(+)-K(+)-2Cl- cotransporter, and, most likely, a nonselective cation channel. Receptor stimulation was not necessary, since elevation of intracellular Ca2+ concentration ([Ca2+]i) by thapsigargin activated the Na+ transporters at equivalent rates. Cell acidification, activation of protein kinase C, cell shrinkage, and other events associated with the rise of [Ca2+]i had little effect on Na+ transport in resting cells. Nevertheless, stimulation of cells in a medium that prevented normal Ca(2+)-induced cell shrinkage prevented activation of all three transport pathways. The block of the activation was not overcome by osmotic shrinkage but was relieved when [Cl-]i was allowed to fall, including conditions in which [Cl-]i fell in the absence of cell shrinkage. Activation of a Na(+)-H+ exchanger, Na(+)-K(+)-2Cl- cotransporter, and nonselective cation channel therefore exhibits a requirement for agonist-induced fall in [Cl-]i. Low [Cl-]i may create a permissive environment for Ca(2+)-dependent activation of multiple Na(+)-transport pathways, providing a mechanism for cross talk that coordinates transport activities of the apical and basolateral membranes in secretory epithelial cells.

Cholecystokinin (CCK) analogs, JMV-180 and OPE, release Ca2+ from intracellular stores and induce oscillations in the concentration of cytosolic Ca2+ ([Ca2+]i), but do not generate a detectable rise in inositol 1,4,5-trisphosphate (InsP3) levels. In contrast, high concentrations of CCK elevate InsP3, as well [Ca2+]i, to a peak which decreases to near basal levels without oscillations. The mechanisms which underlie inhibition of [Ca2+]i oscillations observed with high CCK concentrations are unclear, but are believed to involve a low-affinity CCK receptor state. Alternately, CCK analogs may be weak partial agonists of the phospholipase C pathway, whereas native CCK, as a full agonist of this pathway, stimulates low levels of protein kinase C (PKC) activity. Preincubation of acini with 1 nM 12 O-tetradecanoyl-phorbol 13-acetate (TPA) for 15 min at 37 degrees C did not affect OPE binding to acini, but abolished OPE-induced (at 1 microM) [Ca2+]i oscillations without affecting the initial [Ca2+]i spike. These transformed OPE-induced [Ca2+]i responses mimicked those induced by supramaximal CCK octapeptide (CCK-8) concentrations. Inhibition of [Ca2+]i oscillations by 1 nM TPA was reversed by the PKC inhibitor staurosporine (0.2 microM). After [Ca2+]i oscillations were induced with OPE or low concentrations of CCK-8 (20 pM), 1 nM TPA caused a gradual slowing of oscillation frequency over 15-20 min without affecting [Ca2+]i spike amplitude. In contrast, 1 microM TPA inhibited OPE binding and caused a more generalized inhibition of OPE- and CCK-evoked Ca2+ signals.(ABSTRACT TRUNCATED AT 250 WORDS)

Pancreatic acinar cells, a model cell type for the study of exocytosis in non-excitable cells, were used here to test the hypothesis that molecular mechanisms regulating exocytosis from neuronal and neuroendocrine cells may also be utilized in exocrine cells. Using specific antisera raised against vesicle-associated membrane protein (VAMP) isoforms 1 and 2, we have identified VAMP-2, but not VAMP-1, immunoreactive proteins on rat pancreatic acinar cell secretory (zymogen) granules. This 18-kDa protein co-migrated with rat brain synaptic vesicle VAMP-2. Tetanus toxin (TeTx) light chain caused complete cleavage of this protein, which was prevented by the addition of EDTA. This toxin also inhibited in a dose-dependent manner Ca(2+)-stimulated enzyme secretion by up to approximately 30% in streptolysin O-permeabilized acini. This effect of TeTx on secretion was prevented by the zinc endopeptidase inhibitor captopril or by boiling the toxin. Incomplete inhibition of secretion by TeTx may suggest that TeTx-insensitive or VAMP-2-independent mechanisms also regulate exocytosis. In support, TeTx light chain was without effect on Rab3AL (effector domain peptide of Rab3)-mediated potentiation of Ca(2+)-stimulated secretion. These results indicate that a TeTx-sensitive VAMP-2-like protein on zymogen granules participates in Ca(2+)-regulated pancreatic enzyme secretion and that undefined Rab3AL-activated mechanisms may act independently to regulate exocytosis.

Stimulation of salivary secretion is associated with a rise of [Ca2+]i in acinar cells. We examined the osmotic and ionic consequences of activation of Ca(2+)-dependent K+ and Cl- channels, by simultaneous optical determinations of cell volume and [Ca2+]i, [Cl-]i or [Na+]i during muscarinic stimulation of single salivary acinar cells, using a differential interference contrast (DIC)-fluorescence microscope. Carbachol caused a rapid rise of [Ca2+]i, as well as a substantial cell shrinkage. Despite variability in the level and kinetics of the subsequent sustained phase of the [Ca2+]i response, cell volume was correlated with [Ca2+]i in all cases. Elevated [Ca2+]i was both necessary and sufficient to cause these changes in cell volume. The proposition that changes in cell volume reflected changes in cell solute content was confirmed by simultaneously measuring [Cl-]i and cell volume. Simultaneous determinations of cell volume and [Na+]i indicated that the initial cell shrinkage was due entirely to K+ and Cl- efflux. Subsequent to the initial shrinkage, [Na+]i rose to high levels, primarily due to activation of Na+/H+ exchange. Thus, modulation of ion transport activities under isosmotic conditions results in substantial changes in cell solute content and cell volume. Subsequent to the early Ca(2+)-induced changes in these parameters, other transporters become active, but it is unclear what signals their activation. Cell swelling by osmotic dilution of the bath resulted in compensatory cell shrinkage (RVD) which was sensitive to K+ and Cl- gradients. Nevertheless, a rise of [Ca2+]i was not necessary for RVD. Osmotic shrinkage and/or cell acidification were insufficient to activate Na+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonselective cation (NSC) channels have been identified in the apical membrane of fetal distal lung epithelium (FDLE). However, their physiological role in Na+ transport is uncertain. Because terbutaline, a beta 2-agonist, increases Na+ transport by FDLE, we studied its effect and selected signal transduction mechanisms on NSC channel activity. Using patch-clamp and single-cell imaging techniques, we found that terbutaline activated the NSC channel by 1) increasing its sensitivity to cytosolic Ca2+ concentration ([Ca2+]c) by 100- to 1,000-fold, 2) increasing [Ca2+]c from 35 nM to 3.3 microM, 3) producing a dependency of the NSC channel activity on the cytosolic Cl- concentration ([Cl-]c) at a physiological [Ca2+]c, and 4) inducing a reduction in the [Cl-]c from 45 to 25 mM, which directly activates the beta 2-treated NSC channel. These observations indicate that a beta 2-agonist physiologically activates an amiloride-blockable NSC channel in FDLE through an increase in its sensitivity to [Ca2+]c, resulting in the development of a [Cl-]c dependency at a physiological [Ca2+]c associated with both an increase in [Ca2+]c and a reduction in [Cl-]c. A development of the [Cl-]c dependency and a reduction in [Cl-]c act as a second messenger of the beta-agonist signal transduction pathway in this Na(+)-transporting epithelium.

Fluid secretion by epithelial cells is modulated by agents which activate Cl- channels in the apical membrane. To sustain secretion, Cl- influx across the basolateral membrane must also be accelerated. To examine cellular mechanisms which couple Cl- efflux across the apical membrane to Na(+)-coupled Cl- entry across the basolateral membrane, we employed optical imaging techniques utilizing single rat salivary acinar cells. Na+ influx was negligible in resting cells, but was rapidly increased by carbachol due to activation of a Na(+)-H+ exchanger, Na(+)-K(+)-2Cl- cotransporter and likely a non-selective cation channel. Receptor stimulation was not necessary since elevation of [Ca2+]i by thapsigargin activated the Na+ transporters at equivalent rates. Cell acidification, activation of protein kinase C, and cell shrinkage, other events associated with the rise of [Ca2+]i, had little effect on Na+ transport in resting cells. Nevertheless, stimulation of cells in a medium which prevented normal Ca(2+)-induced cell shrinkage prevented activation of all three pathways. The block of the activation was not overcome by osmotic shrinkage, but was relieved when intracellular Cl- concentration ([Cl-]i) was allowed to fall, including conditions in which [Cl-]i fell in the absence of cell shrinkage. Activation of a Na(+)-H+ exchanger, Na(+)-K(+)-2Cl- cotransporter, and non-selective cation channel therefore exhibit a requirement for agonist-induced fall in [Cl-]i. Low [Cl-]i may create a permissive environment for Ca(2+)-dependent activation of multiple Na+ transport pathways, providing for crosstalk which coordinates transport activities of apical and basolateral membranes in secretory epithelial cells.

The regulation by ATP of Cl- secretion in T84 cells grown on filters was investigated by measuring short-circuit current (Isc = net Cl- secretion). ATP (greater than or equal to 10 microM) added to the basolateral side markedly stimulated Isc both in the presence and absence of forskolin-activated Isc. Fluorescence microscopy of cells loaded with the Ca2+ indicator fura-2 showed that ATP stimulated a transient increase in intracellular free Ca2+ concentration [Ca2+]i. The augmentation of forskolin-stimulated Isc by ATP was at least partly caused by mobilization of Ca2+ from an internal store because prior depletion of the store using ionomycin prevented the response. The activity sequence for stimulation of Isc in the presence of forskolin was adenosine 5'-O-(3-thiotriphosphate) = 5'-adenylylimidodiphosphate (AMP-PNP) greater than ATP greater than ADP greater than AMP, suggesting the presence of a P2 purinergic receptor. Neither beta, gamma-methyleneadenosine 5'-triphosphate nor alpha, beta-methyleneadenosine 5'-triphosphate increased the Isc. Stimulation of Isc by ATP in the absence of forskolin was at least partly due to the breakdown of ATP to AMP and adenosine, which act at P1 receptors to stimulate Isc, since 1) inhibition of the ecto-phosphohydrolase 5'-nucleotidase by alpha, beta-methylene-ADP partially inhibited stimulation of Isc by ATP, 2) the adenosine receptor antagonists caffeine and 8-phenyltheophylline markedly inhibited the ATP-stimulated Isc, and 3) AMP-PNP, a weakly hydrolyzable analogue of ATP, caused a much smaller increase in Isc compared with ATP. Adenosine had no effect on [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

A cAMP-inducible chloride permeability has been detected in mouse fibroblast (L cell) lines upon stable integration of a full-length cDNA encoding the human cystic fibrosis transmembrane conductance regulator (CFTR). As indicated by a Cl--indicator dye, the Cl- permeability of the plasma membrane increases by 10- to 30-fold within 2 min after treatment of the cells with forskolin, an activator of adenylyl cyclase. The properties of the conductance are similar to those described in secretory epithelial cells; the whole-cell current-voltage relationship is linear and there is no evidence of voltage-dependent inactivation or activation. In contrast, this cAMP-dependent Cl- flux is undetectable in the untransfected cells or cells harboring defective cDNA constructs, including one with a phenylalanine deletion at amino acid position 508 (ΔF508), the most common mutation causing cystic fibrosis. These observations are consistent with the hypothesis that the CFTR is a cAMP-dependent Cl- channel. The availability of a heterologous (nonepithelial) cell type expressing the CFTR offers an excellent system to understand the basic mechanisms underlying this CFTR-associated ion permeability and to study the structure and function of the CFTR., link_to_OA_fulltext

The microsomal Ca-ATPase inhibitor thapsigargin induces in rat salivary acinar cells [Ca2+]i oscillations which, though similar to those activated by agonists, are independent of inositol phosphates or inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ stores (Foskett, J. K., Roifman, C., and Wong, D. (1991) J. Biol. Chem. 266, 2778-2782). To examine whether the oscillation mechanism resides in another, thapsigargin- and IP3-insensitive intracellular store, we examined the effects of caffeine and ryanodine, known modulators of Ca2+ release from sarcoplasmic reticulum in excitable cells. Oscillations were induced by caffeine (1-20 mM) in nonoscillating thapsigargin-treated acinar cells, which required the continued presence of caffeine, whereas caffeine was without effect or reduced oscillation amplitude in oscillating cells. Ryanodine (10-50 microM) inhibited oscillations in most of the cells. These results suggest that Ca2+ oscillations in parotid acinar cells are driven by periodic Ca2+ release from an IP3-insensitive Ca2+ store with properties similar to sarcoplasmic reticulum of excitable cells.

The tumor promoter thapsigargin releases Ca2+ from intracellular stores by specific inhibition of microsomal Ca-ATPase activity without inositol phosphate formation. Recent studies of the actions of thapsigargin support the concept that the level of Ca2+ within the inositol (1,4,5)-trisphosphate (IP3)-sensitive intracellular pool regulates the Ca2+ permeability of the plasma membrane. We examined the effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) in single rat parotid cells using digital fluorescence microscopy. In the absence of extracellular Ca2+ (Ca2+o), thapsigargin transiently increased [Ca2+]i. Following the thapsigargin-induced [Ca2+]i transient, carbachol in the continued absence of Ca2+o was unable to raise [Ca2+]i, indicating that thapsigargin mobilizes Ca2+ from the IP3-sensitive store. In the converse experiment, carbachol prevented a rise of [Ca2+]i by thapsigargin, suggesting that the IP3- and thapsigargin-sensitive Ca2+ pools are the same. Depletion of Ca2+ from the IP3-sensitive pool by thapsigargin enhanced plasma membrane Ca2+ permeability. Thapsigargin triggered sustained Ca2+ oscillations in Ca2(+)-containing medium which are highly reminiscent of agonist-induced oscillations in these cells. Carbachol addition rapidly raised IP3 levels during oscillations triggered by thapsigargin but did not elevate [Ca2+]i, indicating that the IP3-sensitive pool remains continuously depleted during [Ca2+]i fluctuations. The results from this study rule out the involvement of the IP3-sensitive pool in the mechanisms involved in thapsigargin-induced (and by analogy, agonist-induced) oscillations in parotid cells.

Differential interference contrast microscopy and low-light-level digital imaging of the fluorescent chloride indicator dye 6-methyl-1-(3-sulfonatopropyl)quinolinium (SPQ) were performed simultaneously in single mammalian salivary gland acinar cells to examine the relationship between cytoplasmic chloride concentration [( Cl-]i) and cell volume during stimulus-secretion coupling. Agonist stimulation of Cl(-)-driven fluid secretion is associated with rapid, Ca2(+)-dependent changes of cell volume, which are temporally coupled to changes of [Cl-]i. The agonist-induced changes in [Cl-]i, if accompanied by cations and water, quantitatively account for the cell volume changes, demonstrating in a single cell that cell volume is determined by cell solute content. Agonist-induced modulation of cell volume appears to be a consequence of the requirement to develop appropriate ion gradients necessary for vectorial salt (and fluid) transport.

Modulation of cytoplasmic free Ca2+ concentration ([Ca2+]i) by receptor-mediated generation of inositol 1,4,5-trisphosphate (InsP3) and activation of its receptor (InsP3R), a Ca2+-release channel in the endoplasmic reticulum, is a ubiquitous signalling mechanism. A fundamental aspect of InsP3-mediated signalling is the graded release of Ca2+ in response to incremental levels of stimuli. Ca2+ release has a transient fast phase, whose rate is proportional to [InsP3], followed by a much slower one even in constant [InsP3]. Many schemes have been proposed to account for quantal Ca2+ release, including the presence of heterogeneous channels and Ca2+ stores with various mechanisms of release termination. Here, we demonstrate that mechanisms intrinsic to the single InsP3R channel can account for quantal Ca2+ release. Patch-clamp electrophysiology of isolated insect Sf9 cell nuclei revealed a consistent and high probability of detecting functional endogenous InsP3R channels, enabling InsP3-induced channel inactivation to be identified as an inevitable consequence of activation, and allowing the average number of activated channels in the membrane patch (NA) to be accurately quantified. InsP3-activated channels invariably inactivated, with average duration of channel activity reduced by high [Ca2+]i and suboptimal [InsP3]. Unexpectedly, NA was found to be a graded function of both [Ca2+]i and [InsP3]. A qualitative model involving Ca2+-induced InsP3R sequestration and inactivation can account for these observations. These results suggest that apparent heterogeneous ligand sensitivity can be generated in a homogeneous population of InsP3R channels, providing a mechanism for graded Ca2+ release that is intrinsic to the InsP3R Ca2+ release channel itself.

A video microscope designed to allow low light level fluorescence imaging of cells during simultaneous high-resolution differential interference contrast (DIC) imaging, without the fluorescence light losses of 60-90% normally associated with this contrast-enhancement technique, is described. Transmitted light for DIC imaging, filtered at greater than 620 nm, passes through standard DIC optical components, (1/4 lambda-plate, polarizer, and Wollaston prism) before illuminating the cells. Transmitted light and fluorescence emission pass through a second Wollaston prism but not through the analyzer, which is repositioned more distally in the optical path. Prisms designed to reflect light out a side port of the microscope to a video camera have been replaced with a dichroic mirror. This mirror reflects fluorescence emission out the side port to a low light-sensitive video camera. The spectrally distinct transmitted light continues through the dichroic mirror to an overhead camera through a polarizer (analyzer), which completes the DIC optical path. The fluorescence and DIC images can be viewed simultaneously on side-by-side video monitors, examined sequentially by an image-processing computer, or examined simultaneously using a video splitter/inserter. The ability to image cells with high resolution simultaneously with low light level fluorescence imaging should find wide applicability whenever it is necessary or desirable to correlate fluorescence intensity or distribution with specific cell structure or function.

Muscarinic stimulation of fluid secretion by mammalian salivary acinar cells is associated with a rise in the level of intracellular free calcium ([Ca2+]i) and activation of a calcium-sensitive potassium (K+) conductance in the basolateral membrane. To test in the intact cell whether the rise of [Ca2+]i precedes activation of the K+ conductance (as expected if Ca2+ is the intracellular messenger mediating this response), [Ca2+]i and membrane voltage were measured simultaneously in carbachol-stimulated rat parotid acinar cells by using fura-2 and an intracellular microelectrode. Unexpectedly, the cells hyperpolarize (indicating activation of the K+ conductance) before fura-2 detectable [Ca2+]i begins to rise. This occurs even in Ca2+-depleted medium where intracellular stores are the only source of mobilized Ca2+. Nevertheless, when the increase in [Ca2+]i was eliminated by loading cells with the Ca2+ chelator bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetate (Me2BAPTA) and stimulating in Ca2+-depleted medium, membrane hyperpolarization was also eliminated, indicating that a rise of [Ca2+] is required for the agonist-induced voltage response. Stimulation of Me2BAPTA-loaded cells in Ca2+-containing medium dramatically accentuates the temporal dissociation between the activation of the K+ conductance and the rise of [Ca2+]i. The data are consistent with the hypothesis that muscarinic stimulation results in a rapid localized increase in [Ca2+]i at the acinar basolateral membrane followed by a somewhat delayed increase in total [Ca2+]i. The localized increase cannot be detected by fura-2 but is sufficient to open the Ca2+-sensitive K+ channels located in the basolateral membrane. We concluded that a receptor-mobilized intracellular store of Ca2+ is localized at or near the basolateral membrane.

The importance of calcium and cellular cytoskeletal elements in the activation or control of volume regulation by epithelial cells was explored in Necturus gallbladder. Gallbladder cells have been previously shown to rapidly readjust their volumes to control size after osmotic perturbation of the mucosal bathing solution. Removal of calcium from the perfusates caused dramatic morphological changes that prevented assessment of the role of extracellular calcium in volume regulation. The regulatory volume increase (RVI) that follows shrinkage of the cell due to perfusion of a hypertonic mannitol solution is insensitive to agents that interfere with cell calcium- or calmodulin-mediated events (quinidine, trifluoperazine) and is not blocked by agents that cause changes in the cytoskeleton (colchicine, cytochalasin B). Osmotically induced cell swelling is followed by regulatory volume decrease (RVD), which is inhibited by agents that interfere with calcium-dependent processes (quinidine, trifluoperazine) and by the microfilament inhibitor, cytochalasin B. These results indicate that RVD depends on calcium, calmodulin, and an intact microfilament network, whereas RVI is independent of these factors.

Previous work has demonstrated that after osmotic shrinkage of Necturus gallbladder epithelial cells, their volumes are restored to control levels despite the continued presence of the hyperosmotic medium. It has been proposed that activation of parallel neutral Na+-H+ and Cl--HCO-3 exchangers in the apical membrane is necessary for regulatory volume increase. As an independent technique to determine whether and for how long ion flux through the anion exchanger is actually enhanced by exposure to hypertonicity, fluorescence measurements of N-(2-aminoethylsulfonate)-7-nitrobenz-2-oxa-1,3-diazole (NBD-taurine), a substrate of the anion exchanger in red blood cells, have been made in intact Necturus gallbladder. The cells were loaded with the dye by incubation. The tissue was perfused in a miniature chamber placed on the stage of a microscope and viewed with high-magnification optics combined with video. Fluorescence was monitored at frequent intervals with a photomultiplier tube, and transmittance of the tissue to the laser excitation light was monitored with a photodiode. The epithelium was simultaneously observed with transmitted light to control for changes in focus or lateral movement. Exposure of the tissue to a mucosal medium made hypertonic by the addition of mannitol transiently enhanced the efflux of NBD-taurine from the cells in approximately 70% of the tissues examined. In the presence of the anion-exchange inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS, 100 microM), hypertonicity enhanced NBD-taurine efflux in only 14% of the preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of prolactin on transport properties of opercular membranes from seawater-adapted tilapia, Sarotherodon mossambicus, have been examined. These membranes are high conductance (average Gt approximately 4 mS.cm-2) tissues with short-circuit currents (I) equal to net chloride secretion. Despite high Gt, nonlinear current-voltage relationships suggest that opercular membranes cannot be classified as "leaky" tissues. Variability among membranes is reflected in a linear relationship between I and Gt with a slope equal to 26 mV and the zero-current Gt intercept equal to 0.45 mS.cm-2. Prolactin injections decrease I and Gt in a dose-dependent manner. Phosphodiesterase inhibition, without effect on I in untreated fish, often partially reverses these prolactin effects. Gt-I data from prolactin-treated fish yield a slope of 18 mV and a Gt intercept of 0.10 mS.cm-2. The effects of prolactin are discussed in terms of conventional equivalent circuit analysis. Discrepancies between predictions based on this model and the actual data indicate that an alternative interpretation, based on a heterogeneous cell population, is more accurate. Analysis of this circuit suggests that the ratio of paracellular to active transport pathway conductances associated with chloride cells is constant and that differences in Gt and I are due to parallel changes in these conductances. Prolactin may effectively "remove" chloride cells from these membranes as well as inhibit (reversible by elevated cellular cAMP levels) active transport pathway conductance of remaining cells.

Cell volume determinations and electrophysiological measurements have been made in an attempt to determine if mitochondria-rich (MR) cells are localized pathways for conductive movements of Cl across frog skin epithelium. Determinations of cell volume with video microscope techniques during transepithelial passage of current showed that most MR cells swell when the tissue is voltage clamped to serosa-positive voltages. Voltage-induced cell swelling was eliminated when Cl was removed from the mucosal bath solution. Using a modified vibrating probe technique, it was possible to electrically localize a conductance specifically to some MR cells in some tissues. These data are evidence supporting the idea that MR cells are pathways for conductive movements of Cl through frog skin epithelium.

Teleost fish osmoregulation is largely the result of integrated transport activities of the gill, gut and renal system. The basic ‘epithelial fabric’ in each of these tissues is adapted to provide the appropriate transport mechanisms depending upon whether the fish is in fresh water or sea water. Net NaCl transport by the branchial epithelium reverses direction when euryhaline species migrate between the two media, providing a useful focus in experiments designed to elucidate mechanisms of differentiation and integration of transport function. Isolated opercular membranes and skins from certain seawater-adapted species are good models to study branchial salt extrusion mechanisms. These heterogeneous tissues generate short-circuit currents equal to net chloride secretion. The vibrating probe technique has allowed localization of all current and almost all conductance to the apical crypt of chloride cells. Area-specific surface current and conductance of chloride cells are 18mAcm−2 and 580mScm−2 (1·7Ωcm2), ranking them as one of the most actively transporting and conductive cells known. There is no net sodium transport under short-circuit conditions but the chloride secretion process is sodium-dependent and ouabain and ‘loop’-diuretic sensitive. Sodium fluxes through chloride cells are large (PNa = 5·2 × Ω 10−4cms−1) and appear passive and rate-limited by a single barrier. A link may exist between the active transport and leak pathways since sodium fluxes always account for 50% of chloride cell conductance. The sodium pathway is probably the chloride cell-accessory cell tight junction, although this is still unresolved. Chloride secretion can be rapidly modulated by several hormones, including catecholamines, somatostatin, glucagon, vasoactive intestinal polypeptide and urotensins I and II. Regulation by these hormones may be by rapid alterations of cellular cAMP levels. Differentiation of chloride cells and chloride secretion may be controlled by cortisol and prolactin. Cortisol stimulates chloride cell proliferation and differentiation and appears to interact with NaCl to initiate salt secretion. Prolactin appears to cause chloride cell dedifferentiation by reducing both the active-transport and leak pathways proportionately. Prolactin and cortisol also affect epithelial cell proliferation and differentiation in the other osmoregulatory tissues in fish, suggesting that these hormones are primary agents in the integration of transport activities to achieve whole animal osmoregulation.