Regulatory interaction of ATP Na+ and Cl- in the turnover cycle of the NaK2Cl cotransporter

To probe the mechanism by which intracellular ATP, Na+, and Cl- influence the activity of the NaK2Cl cotransporter, we measured bumetanide-sensitive (BS) 86Rb fluxes in the osteosarcoma cell line UMR- 106-01. Under physiological gradients of Na+, K+, and Cl-, depleting cellular ATP by incubation with deoxyglucose and antimycin A (DOG/AA) for 20 min at 37 degrees C reduced BS 86Rb uptake from 6 to 1 nmol/mg protein per min. Similar incubation with 0.5 mM ouabain to inhibit the Na+ pump had no effect on the uptake, excluding the possibility that DOG/AA inhibited the uptake by modifying the cellular Na+ and K+ gradients. Loading the cells with Na+ and depleting them of K+ by a 2-3- h incubation with ouabain or DOG/AA increased the rate of BS 86Rb uptake to approximately 12 nmol/mg protein per min. The unidirectional BS 86Rb influx into control cells was approximately 10 times faster than the unidirectional BS 86Rb efflux. On the other hand, at steady state the unidirectional BS 86Rb influx and efflux in ouabain-treated cells were similar, suggesting that most of the BS 86Rb uptake into the ouabain-treated cells is due to K+/K+ exchange. The entire BS 86Rb uptake into ouabain-treated cells was insensitive to depletion of cellular ATP. However, the influx could be converted to ATP-sensitive influx by reducing cellular Cl- and/or Na+ in ouabain-treated cells to impose conditions for net uptake of the ions. The BS 86Rb uptake in ouabain-treated cells required the presence of Na+, K+, and Cl- in the extracellular medium. Thus, loading the cells with Na+ induced rapid 86Rb (K+) influx and efflux which, unlike net uptake, were insensitive to cellular ATP. Therefore, we suggest that ATP regulates a step in the turnover cycle of the cotransporter that is required for net but not K+/K+ exchange fluxes. Depleting control cells of Cl- increased BS 86Rb uptake from medium-containing physiological Na+ and K+ concentrations from 6 to approximately 15 nmol/mg protein per min. The uptake was blocked by depletion of cellular ATP with DOG/AA and required the presence of all three ions in the external medium. Thus, intracellular Cl- appears to influence net uptake by the cotransporter. Depletion of intracellular Na+ was as effective as depletion of Cl- in stimulating BS 86Rb uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of chloride deficiency on the pancreatic B-cell response to acetylcholine

Muscarinic stimulation of pancreatic B-cells markedly amplifies insulin secretion through complex mechanisms which involve changes in membrane potential and ionic fluxes. In this study, normal mouse islets were used to evaluate the role of Cl- ions in these effects of acetylcholine (ACh). Whatever the concentration of glucose, the rate of 36Cl- efflux from islet cells was unaffected by ACh. Replacement of Cl- by impermeant isethionate in a medium containing 15 mM glucose did not affect, or only slightly decreased, the ability of ACh to depolarize the B-cell membrane and increase electrical activity, to accelerate 45Ca2+ and 86Rb+ efflux from islet cells, and to amplify insulin release. In the absence of extracellular Ca2+, a high concentration of ACh (100 microM) mobilized intracellular Ca2+ and caused a transient release of insulin and a sustained acceleration of 86Rb+ efflux. None of these effects was affected by Cl- omission or by addition of furosemide, a blocker of the Na+, K+, 2Cl- cotransport. Isethionate substitution for Cl- in a medium containing a nonstimulatory concentration of glucose (3 mM) barely reduced the depolarization of B-cells by ACh, but inhibited the concomitant increase in 86Rb+ efflux. We have no explanation for the latter effect that was not mimicked by furosemide. In conclusion, ACh stimulation of pancreatic B-cells, unlike that of exocrine acinar cells, is largely independent of Cl- and is insensitive to furosemide. The acceleration of ionic fluxes produced by ACh does not involve the Na+, K+, 2Cl- cotransport system.     

Mechanism of the excitatory Cl- response in mouse olfactory receptor neurons

In vertebrate olfactory receptor neurons (ORNs), the odorant-triggered receptor current flows through two distinct ion channels on the sensory cilia: Ca2+ influx through a cyclic nucleotide-gated (CNG) channel followed by Cl- efflux through a Ca2+-activated anion channel. The excitatory Cl- current amplifies the small CNG current and crucially depends on a high intracellular Cl- concentration. We show here that a (Na+)-(K+)-(2Cl-) cotransporter, NKCC1, is required for this Cl- current, in that ORNs deficient in Nkcc1 or incubated with an NKCC blocker (bumetanide) lack the Cl- current. Surprisingly, immunocytochemistry indicates that NKCC1 is located on the somata and dendrites of ORNs rather than the cilia, where transduction occurs. This topography is remarkably similar to the situation in secretory epithelial cells, where basolateral Cl- uptake and apical Cl- efflux facilitate transepithelial fluid movement. Thus, a single functional architecture serves two entirely different purposes, probably underscoring the epithelial origin of the ORNs.     

Species-dependent differences in the influence of ionic strength on potassium transport of erythrocytes. The role of membrane fluidity and Ca2+

The passive Rb+ (K+) efflux from erythrocytes of seven mammalian species was investigated in solutions of physiological and low ionic strength. Furthermore the fluidity of the erythrocyte membrane in the same solutions was estimated by measuring the ESR order parameter. The rate constant of Rb+ (K+) efflux in solution of high ionic strength could be correlated with the order parameter obtained and with the mean number of double bonds to the membrane phospholipid fatty acids. The same relationships could be observed for the low ionic strength solutions if the values for human erythrocytes were excluded. The appearance of Na+, K+, Cl- cotransport to a significant extent, only in human erythrocytes, was supposed to be the reason for this different behaviour of human red blood cells. It was demonstrated that the strong increase of the Rb+ (K+) efflux rate constant for human erythrocytes in low ionic strength solution is not due to Ca2+, as quinine treatment and replacement of all external potassium, both inhibiting the Ca2(+)-induced K+ efflux, did not abolish the increase of (Rb+) K+ efflux in solutions of low ionic strength.     

Significance of Ca2+, Rb+ fluxes, of cAMP and cGMP for the CCK8-modulated insulin release

In rat pancreatic islets the effects of cholecystokinin-8 (CCK8) on glucose-mediated insulin release, 45Ca2+ net uptake, 45Ca2+ efflux, 86Rb+ efflux, cAMP- and cGMP levels were studied. In the presence of a substimulatory glucose concentration (3 mM) CCK8 concentrations of up to 1 microM had no effect on insulin release, but CCK8 at 10 nM potentiated the stimulatory effect of glucose (11.1 mM). 10 nM CCK8 enhanced glucose-stimulated 45Ca2+ net uptake but was ineffective at substimulatory glucose levels. CCK8 had no effect on cAMP and cGMP levels in the presence of 11.1 mM glucose, CCK8 increased 86Rb+ (a measure of K+) in the presence of both 3 and 11.1 mM glucose. This effect was abolished when Ca2+ was omitted from the perifusion medium. CCK8 did not alter glucose (11.1 mM)-stimulated 45Ca2+ efflux rate. These data indicate that (1) CCK8 potentiates glucose-stimulated insulin secretion possibly via an effect on Ca2+ uptake, 2) by affecting Ca2+ uptake, CCK8 enhances K+ efflux, and 3) CCK8 does not mediate its effect via cAMP or cGMP. With respect to 86Rb+ efflux the mechanism of CCK8 action appears to be different from that of glucose. When the mechanism of CCK action on islets is compared with that on exocrine pancreas (data from others) there are similarities (importance of Ca2+ uptake and non-importance of cAMP and cGMP).     

Effects of extracellular ATP on ion transport systems and [Ca2+]i in rat parotid acinar cells. Comparison with the muscarinic agonist carbachol

The effects of extracellular ATP on ion fluxes and the intracellular free Ca2+ concentration ([Ca2+]i) were examined using a suspension of rat parotid acinar cells and were contrasted with the effects of the muscarinic agonist carbachol. Although ATP and carbachol both rapidly increased [Ca2+]i about threefold above the resting level (200-250 nM), the effect of ATP was due primarily to an influx of Ca2+ across the plasma membrane, while the initial response to carbachol was due to a release of Ca2+ from intracellular stores. Within 10 s, ATP (1 mM) and carbachol (20 microM) reduced the cellular Cl- content by 39-50% and cell volume by 15-25%. Both stimuli reduced the cytosolic K+ content by 57-65%, but there were marked differences in the rate and pattern of net K+ movement as well as the effects of K+ channel inhibitors on the effluxes initiated by the two stimuli. The maximum rate of the ATP-stimulated K+ efflux (approximately 2,200 nmol K+/mg protein per min) was about two-thirds that of the carbachol-initiated efflux rate, and was reduced by approximately 30% (vs. 60% for the carbachol-stimulated K+ efflux) by TEA (tetraethylammonium), an inhibitor of the large conductance (BK) K+ channel. Charybdotoxin, another K+ channel blocker, was markedly more effective than TEA on the effects of both agonists, and reduced the rate of K+ efflux initiated by both ATP and carbachol by approximately 80%. The removal of extracellular Ca2+ reduced the ATP- and the carbachol-stimulated rates of K+ efflux by 55 and 17%, respectively. The rate of K+ efflux initiated by either agonist was reduced by 78-95% in cells that were loaded with BAPTA to slow the elevation of [Ca2+]i. These results indicated that ATP and carbachol stimulated the efflux of K+ through multiple types of K(+)-permeable channels, and demonstrated that the relative proportion of efflux through the different pathways was different for the two stimuli. ATP and carbachol also stimulated the rapid entry of Na+ into the parotid cell, and elevated the intracellular Na+ content to 4.4 and 2.6 times the normal level, respectively. The rate of Na+ entry through Na(+)-K(+)-2Cl- cotransport and Na(+)-H+ exchange was similar whether stimulated by ATP, carbachol, or ionomycin, and uptake through these two carrier-mediated transporters accounted for 50% of the ATP-promoted Na+ influx. The remainder may be due to a nonselective cation channel and an ATP-gated cation channel that is also permeable to Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

The response of chloride transport to cyclic AMP, calcium and hypotonic shock in normal and cystic fibrosis fibroblasts

It has widely been established that Cl- transport is defective in cystic fibrosis fibroblasts. In the present study, the effect of elevation of intracellular concentration of cyclic AMP and calcium on the efflux of Cl- from human fibroblasts has been investigated. Cyclic AMP analogs (8-bromo cAMP and dibutyryl cAMP) and a beta agonist (isoproterenol) induced only a weak stimulation (5-10%) of Cl- efflux. Conversely, elevation of cytoplasmic calcium concentration produced by addition of the Ca2+ ionophore A23187 in the efflux medium, did not affect Cl- efflux. Our data indicate that the response of Cl- efflux to elevation of cAMP and calcium is similar in normal and cystic fibrosis fibroblasts. Exposure to hypotonic medium induced a significant stimulation of Cl- efflux in fibroblasts from both normal and cystic fibrosis individuals. Substitution of Cl- in the medium by gluconate and the subsequent addition of furosemide did not inhibit the effect of hypotonicity, indicating the involvement of a conductive pathway for Cl- transport, which was insensitive to oligomicin C.     

Nitrendipine is a potent inhibitor of the Ca(2+)-activated K+ channel of human erythrocytes.

Nitrendipine, a classical blocker of L-type Ca2+ channels, is shown to be a potent inhibitor of the Ca(2+)-activated K+ channel of human erythrocytes. In erythrocytes suspended in a solution with physiological Na+ and K+ concentrations and in which the channel was activated using the Ca2+ ionophore ionomycin, nitrendipine inhibited K+(86Rb+) influx with an I50 of around 130 nM. Similar results were obtained for K+(86Rb+) efflux, and for K+(86Rb+) influx into cells suspended in a high-K+ medium.     

Presence of a Ca2+-dependent K+ channel in brown adipocytes. Possible role in maintenance of alpha 1-adrenergic stimulation

We have previously demonstrated mobilization of Ca2+ in and efflux of Rb+ (K+) from isolated hamster brown adipocytes as a consequence of norepinephrine stimulation. We have now investigated the adrenoceptor subtype specificity of these responses and found them both to be of the alpha 1-subtype. Further, we have found that the Rb+ (K+) efflux was dependent upon a primary Ca2+ mobilization event in response to the alpha 1-adrenergic stimulation, since the Rb+ efflux could also be demonstrated by the addition of the Ca2+ ionophore A23187 to the cells. The norepinephrine- and A23187-stimulated Rb+ effluxes were both inhibited by the Ca2+-dependent K+-channel blocker apamin. Apamin also significantly attenuated Ca2+ mobilization in cells in response to a submaximal concentration of norepinephrine. We conclude that alpha 1-adrenergic stimulation of brown fat cells leads to a mobilization of intracellular Ca2+ which, in itself or via other mechanisms, leads to an increase in cytosolic Ca2+ concentration which, in turn, activates a Ca2+-dependent K+ channel, leading to a K+ release from these cells. A possible role for this channel to sustain and augment the response to alpha 1-adrenergic stimulation is discussed.     

Glucose-, calcium- and concentration-dependence of acetylcholine stimulation of insulin release and ionic fluxes in mouse islets.

Mouse islets were used to define the glucose-dependence and extracellular Ca2+ requirement of muscarinic stimulation of pancreatic beta-cells. In the presence of a stimulatory concentration of glucose (10 mM) and of Ca2+, acetylcholine (0.1-100 microM) accelerated 3H efflux from islets preloaded with myo-[3H]inositol. It also stimulated 45Ca2+ influx and efflux, 86Rb+ efflux and insulin release. In the absence of Ca2+, only 10-100 microM-acetylcholine mobilized enough intracellular Ca2+ to trigger an early but brief peak of insulin release. At a non-stimulatory concentration of glucose (3 mM), 1 microM- and 100 microM-acetylcholine increased 45Ca2+ and 86Rb+ efflux in the presence and absence of extracellular Ca2+. However, only 100 microM-acetylcholine marginally increased 45Ca2+ influx and caused a small, delayed, stimulation of insulin release, which was abolished by omission of Ca2+. At a maximally effective concentration of glucose (30 mM), 1 microM- and 100 microM-acetylcholine increased 45Ca2+ influx and efflux only slightly, but markedly amplified insulin release. Again, only 100 microM-acetylcholine mobilized enough Ca2+ to trigger a peak of insulin release in the absence of Ca2+. The results thus show that only high concentrations of acetylcholine (greater than or equal to 10 microM) can induce release at low glucose or in a Ca2+-free medium. beta-Cells exhibit their highest sensitivity to acetylcholine in the presence of Ca2+ and stimulatory glucose. Under these physiological conditions, the large amplification of insulin release appears to be the result of combined effects of the neurotransmitter on Ca2+ influx, on intracellular Ca2+ stores and on the efficiency with which Ca2+ activates the releasing machinery.     

Calcium-activated chloride conductance of lactotrophs: Comparison of activation in normal and tumoral cells during thyrotropin-releasing-hormone stimulation

We studied a chloride (Cl-) conductance activated by calcium (Ca2+) in normal rat lactotrophs and compared its activation during TRH stimulation in normal rat lactotrophs and in GH3 tumoral lactosomatotrophs cells, using the whole-cell configuration of the patch-clamp technique. The Cl- specificity of the conductance was assessed by manipulation of internal and external Cl- concentrations. The reversal potentials were in agreement with those predicted by the Nernst equation. Ca2+ ionophore A23187 and membrane depolarizations activated the Cl- conductance. However, a feedback effect of Cl- gradient modifications on Ca2+ movements was also observed in normal lactotrophs. In the latter, TRH (100 nM) mobilization of intracellular Ca2+ activated this Cl- conductance together with the potassium (K+) conductance when both ions were present in the intracellular medium (IM) or alone when K+ was absent. Chloride conductance was not activated in the GH3 cells, where mobilization of intracellular Ca2+ by TRH (100 nM) activated only Ca2(+)-dependent K+ conductance. It seems likely that the activation of Cl- conductance in these two different cell types involves different mechanisms.     

Properties of the apamin-sensitive Ca2+-activated K+ channel in PC12 pheochromocytoma cells which hyper-produce the apamin receptor

Undifferentiated PC12 cell produce high levels of apamin receptors (measured with 125I-apamin) after 7 days in culture. These levels are at least 50 times higher than those found in other cellular types which are also known to have apamin receptors and apamin-sensitive Ca2+-activated K+ channels in their membranes. Treatment of undifferentiated PC12 cells with nerve growth factor maintains these cells in a state having a low level (10 times less after 7 days of culture) of apamin receptors. Ca2+ injection into PC12 cells with the calcium ionophore A23187 has been used to monitor the activity of the Ca2+-activated K+ channel following 86Rb+ efflux. A large component of this Ca2+-activated 86Rb+ efflux is inhibited by apamin. Half-maximum inhibition by apamin of both 86Rb+ efflux and 125I-apamin binding was observed at 240 pM apamin. Another component of 86Rb+ efflux is due to another type of Ca2+-activated K+ channel which is resistant to apamin and sensitive to tetraethylammonium. The Ca2+ channel activator Bay K8644 also triggers an apamin-sensitive Ca2+-dependent 86Rb+ efflux. Bay K8644 has been used to analyze the internal Ca2+ concentration dependence of the apamin-sensitive channel activity. Under normal conditions, the internal Ca2+ concentration is 109 +/- 17 nM, and the apamin-sensitive channel is not activated. The channel is fully activated at an internal Ca2+ concentration of 320 +/- 20 nM.     

Differential coupling of alpha7 and non-alpha7 nicotinic acetylcholine receptors to calcium-induced calcium release and voltage-operated calcium channels in PC12 cells

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that can modulate various neuronal processes by altering intracellular Ca(2+) levels. Following nAChR stimulation Ca(2+) can enter cells either directly, through the intrinsic ion channel, or indirectly following voltage-operated Ca(2+) channel (VOCC) activation; Ca(2+) levels can subsequently be amplified via Ca(2+)-induced Ca(2+) release from intracellular stores. We have used subtype-selective nAChR agonists to investigate the Ca(2+) sources contributing to alpha7 and non-alpha7 nAChR-mediated increases in intracellular Ca(2+) in PC12 cells. Application of the alpha7 nAChR positive allosteric modulator PNU 120596 (10 mum), in conjunction with the alpha7 nAChR agonist, compound A [(R)-N-(1-azabicyclo[2.2.2]oct-3-yl)(5-(2-pyridyl)thiophene-2-carboxamide), 10 nm], produces a rapid increase in fluo-3 fluorescence that is prevented by the selective alpha7 nAChR antagonist alpha-bungarotoxin. The non-alpha7 nAChR agonist 5-Iodo-A-85380 produces alpha-bungarotoxin-insensitive increases in intracellular Ca(2+) (EC(50) = 11.2 mum). Using these selective agonists or KCl in conjunction with general and selective VOCC inhibitors, we demonstrate that the primary route of Ca(2+) entry following either non-alpha7 nAChR activation or KCl stimulation is via L-type VOCCs. In contrast, the alpha7 nAChR-mediated response is unaffected by VOCC blockers but is inhibited by modulators of intracellular Ca(2+) stores. These results indicate that alpha7 and non-alpha7 nAChRs are differentially coupled to Ca(2+)-induced Ca(2+) release and VOCCs, respectively.     

Activation of the plasma membrane Ca2+ pump during agonist stimulation of pancreatic acini.

The role of internal stores and plasma membrane Ca2+ pumps in controlling [Ca2+]i during agonist stimulation and their regulation by agonists are not well understood. We report here measurements of intracellular ([Ca2+]i) and extracellular ([Ca2+]o) Ca2+ concentrations in agonist-stimulated pancreatic acini in an effort to directly address these questions. Stimulation of acini suspended in Ca(2+)-free or Ca(2+)-containing medium with Ca2+ mobilizing agonists resulted in a typical transient increase in [Ca2+]i. Thapsigargin, a specific inhibitor of internal Ca2+ pumps, inhibited the rate of [Ca2+]i reduction after agonist stimulation by approximately 40%. Under the same conditions, thapsigargin had no effect on the rate of the unidirectional Ca2+ efflux across the plasma membrane as revealed by measurements of [Ca2+]o. These findings suggest that internal Ca2+ pumps actively remove Ca2+ from the cytosol during continued agonist stimulation. The correlation between the reduction in [Ca2+]i and the increase in [Ca2+]o showed that Ca2+ efflux from cells stimulated with agonist and thapsigargin represent Ca2+ efflux across the plasma membrane. Inhibition of cells exposed to agonist and thapsigargin with a specific antagonist sharply reduced the rates of the [Ca2+]i decrease and the accompanied [Ca2+]o increase. Hence, at comparable [Ca2+]i, Ca2+ efflux from stimulated cells was about 3-fold faster than that from resting cells, indicating that agonists directly activate the plasma membrane Ca2+ pump. To study the role of [Ca2+]i increase in plasma membrane Ca2+ pump activation the acini were loaded with 1,2-bis-(2-aminophenoxyethane-N,N,N',N')-tetraacetic acid (BAPTA), and [Ca2+]o was measured during agonist stimulation. Surprisingly, although BAPTA completely prevented the increase in [Ca2+]i, Ca2+ efflux rate was reduced by only 34%. These findings provide the first evidence for Ca(2+)-independent activation of the plasma membrane Ca2+ pump by Ca2+ mobilizing agonists.     

Characterization of increased K+ permeability associated with the stimulation of receptors for immunoglobulin E on rat basophilic leukemia cells.

Aggregation of immunoglobulin E-receptor complexes on the surface of rat basophilic leukemia cells stimulates an increase in plasma membrane K+ permeability that is monitored as an increase in the rate of efflux of preloaded 86Rb+. A major component of this stimulated 86Rb+ efflux appears to be due to a Ca(2+)-activated K+ channel because it is inhibited by quinidine in parallel with the inhibition of degranulation and membrane potential repolarization, it is blocked by 0.1 mM La3+, and it is dependent on external Ca2+. Depolarization of the plasma membrane by carbonyl cyanide 3-chlorophenylhydrazone inhibits stimulated Ca2+ influx and prevents antigen-induced 86Rb+ efflux, and increased external Ca2+ partially restores 86Rb+ efflux under these conditions. In addition, potentiation of antigen-stimulated Ca2+ influx by pretreatment with cholera toxin increases the initial rate of stimulated 86Rb+ efflux. Another component of antigen-stimulated K+ efflux appears to be mediated by a guanine nucleotide-binding protein because pretreatment of rat basophilic leukemia cells with pertussis toxin decreases the initial rate of antigen-stimulated 86Rb+ efflux to 40% of that for the untreated cells. Stimulated 86Rb+ efflux is also observed when ionomycin is used to increase cytoplasmic Ca2+ and to trigger membrane depolarization. The efflux stimulated by ionomycin is inhibited by quinidine but not by pertussis toxin pretreatment; thus, it appears to occur through the Ca(2+)-activated K+ efflux pathway. It is proposed that these K+ efflux pathways serve to sustain the Ca2+ influx that is necessary for receptor-mediated triggering of cellular degranulation.     

Somatostatin inhibits insulin secretion by a G-protein-mediated decrease in Ca2+ entry through voltage-dependent Ca2+ channels in the beta cell.

We tested the hypothesis that somatostatin (SRIF) inhibits insulin secretion from an SV40 transformed hamster beta cell line (HIT cells) by an effect on the voltage-dependent Ca2+ channels and examined whether G-proteins were involved in the process. Ca2+ currents were recorded by the whole cell patch-clamp method, the free cytosolic calcium, [Ca2+]i, was monitored in HIT cells by fura-2, and cAMP and insulin secretion were measured by radioimmunoassay. SRIF decreased Ca2+ currents, [Ca2+]i, and basal insulin secretion in a dose-dependent manner over the range of 10(-12)-10(-7)M. The increase in [Ca2+]i and insulin secretion induced by either depolarization with K+ (15 mM) or by the Ca2+ channel agonist, Bay K 8644 (1 microM) was attenuated by SRIF in a dose-dependent manner over the same range of 10(-12)-10(-7) M. the half-maximal inhibitory concentrations (IC50) for SRIF inhibition of insulin secretion were 8.6 X 10(-12) M and 8.3 X 10(-11) M for K+ and Bay K 8644-stimulated secretion and 1 X 10(-10) M and 2.9 X 10(-10) M for the SRIF inhibition of the K+ and Bay K 8644-induced rise in [Ca2+]i, respectively. SRIF also attenuated the rise in [Ca2+]i induced by the cAMP-elevating agent, isobutylmethylxanthine (1 mM) in the presence of glucose. Bay K 8644, K+ and SRIF had no significant effects on cAMP levels and SRIF had no effects on adenylyl cyclase activity at concentrations lower than 1 microM. SRIF (100 nM) did not change K+ efflux (measured by 86Rb+) through ATP-sensitive K+ channels in HIT cells. SRIF (up to 1 microM) had no significant effect on membrane potential measured by bisoxonol fluorescence. Pretreatment of the HIT cells with pertussis toxin (0.1 microgram/ml) overnight abolished the effects of SRIF on Ca2+ currents, [Ca2+]i and insulin secretion implying a G-protein dependence in SRIF's actions. Thus, one mechanism by which SRIF decreases insulin secretion is by inhibiting Ca2+ influx through voltage-dependent Ca2+ channels, an action mediated through a pertussis toxin-sensitive G-protein.     

Agonist-specific regulation of [Na+]i in pancreatic acinar cells

In a companion paper (Zhao, H., and S. Muallem. 1995), we describe the relationship between the major Na+,K+, and Cl- transporters in resting pancreatic acinar cells. The present study evaluated the role of the different transporters in regulating [Na+]i and electrolyte secretion during agonist stimulation. Cell stimulation increased [Na+]i and 86Rb influx in an agonist-specific manner. Ca(2+)-mobilizing agonists, such as carbachol and cholecystokinin, activated Na+ influx by a tetraethylammonium-sensitive channel and the Na+/H+ exchanger to rapidly increase [Na+]i from approximately 11.7 mM to between 34 and 39 mM. As a consequence, the NaK2Cl cotransporter was largely inhibited and the activity of the Na+ pump increased to mediate most of the 86Rb(K+) uptake into the cells. Secretin, which increases cAMP, activated the NaK2Cl cotransporter and the Na+/H+ exchanger to slowly increase [Na+]i from approximately 11.7 mM to an average of 24.6 mM. Accordingly, secretin increased total 86Rb uptake more than the Ca(2+)- mobilizing agonists and the apparent coupling between the NaK2Cl cotransport and the Na+ pump. All the effects of secretin could be attributed to an increase in cAMP, since forskolin affected [Na+]i and 86Rb fluxes similar to secretin. The signaling pathways mediating the effects of the Ca(2+)-mobilizing agonists were less clear. Although an increase in [Ca2+]i was required, it was not sufficient to account for the effect of the agonists. Activation of protein kinase C stimulated the NaK2Cl cotransporter to increase [Na+]i and 86Rb fluxes without preventing the inhibition of the cotransporter by Ca(2+)-mobilizing agonists. The effects of the agonists were not mediated by changes in cell volume, since cell swelling and shrinkage did not reproduce the effect of the agonists on [Na+]i and 86Rb fluxes. The overall findings of the relationships between the various Na+,K+, and Cl- transporters in resting and stimulated pancreatic acinar cells are discussed in terms of possible models of fluid and electrolyte secretion by these cells.     

Regulatory changes in the K+, Cl- and water contents of HeLa cells incubated in an isosmotic high K(+)-medium

HeLa cells had their normal medium replaced by an isosmotic medium containing 80 mM K+, 70 mM Na+ and 100 microM ouabain. The cellular contents of K+ first increased and then decreased to the original values, that is, the cells showed a regulatory decrease (RVD) in size. The initial increase was not inhibited by various agents except by substitution of medium Cl- with gluconate. In contrast, the regulatory decrease was inhibited strongly by addition of either 1 mM quinine, 10 microM BAPTA-AM without medium Ca2+, or 0.5 mM DIDS, and partly by either 1 mM EGTA without medium Ca2+, 10 microM trifluoperazine, or substitution of medium Cl- with NO3-. Addition of DIDS to the NO3(-)-substituted medium further suppressed the K+ loss but the effect was incomplete. Intracellular Ca2+ showed a transient increase after the medium replacement. These results suggest that the initial increase in cell K+ is a phenomenon related to osmotic water movement toward Donnan equilibrium, whereas the regulatory K+ decrease is caused by K+ efflux through Ca(2+)-dependent K+ channels. The K+ decrease induced a decrease in cellular water, i.e., RVD. The K+ efflux may be more selectively associated with Cl- efflux through DIDS-sensitive channels than the efflux of other anions.