Chloride channels in the luminal membrane of rat pancreatic acini

 Pancreatic acini secrete Na⁺, Cl^–and H₂O in response to secretagogues such as acetylcholine. Cl^–channels in the luminal membrane are a prerequisite for this secretion. The properties of the corresponding conductance have previously been examined using whole-cell recordings. The present study attempts to examine the properties of the single channels in cell-attached and cell-free excised patches from the luminal membrane. To this end the pipettes were filled with an N-methyl-D-glucamine (NMDG⁺) chloride/gluconate solution. The voltage-clamp range was chosen to be pipette positive (cell negative, –60 to –130 mV) in order to increase the driving force for outward Cl^–currents. Under resting conditions cell attached luminal patches had very few single-channel currents (12 out of 45 experiments). Their incidence was sharply increased by carbachol (CCH, 1 μmol/l) in 41 out of 45 experiments. The single-channel conductance of these channels was 1.97 ± 0.05 pS. The properties of these channels in excised patches were examined further: their single-channel conductance was 2.2 ± 0.07 pS (n = 59) and their conductance selectivity was I^– > Br^– > Cl^– >> gluconate. None of the typical Cl^–channel blockers (DIDS, NPPB, glibenclamide 100 μmol/l) blocked these channels. It is concluded that the luminal membrane of the rat pancreatic acinus possesses Cl^–channels with very low conductance which are activated by carbachol. Received: 31 January 1997 / Received after revision: 26 February 1997 / Accepted: 5 March 1997     

Simultaneous presence of cAMP and cGMP exert a co-ordinated inhibitory effect on the agonist-evoked Ca2+ signal in pancreatic acinar cells

The stimulation of the pancreatic acinar cells by physiological secretagogues, such as acetycholine (ACh), activates a well-established intracellular signalling pathway, which involves the generation of Inositol 1,4,5-trisphosphate (InsP₃) and the release of Ca²⁺ from intracellular stores. Caffeine, which inhibits this agonist-evoked Ca²⁺ response reversibly and competitively also blocks the Ca²⁺ signal generated by the non-specific activation of the membrane guanine nucleotide-binding proteins (G-proteins). Removal of caffeine is associated with an increase of intracellular [Ca²⁺] ([Ca²⁺]_i) and the spatial and temporal characteristics of this Ca²⁺ signal are identical to those of the signal generated by the initial agonist stimulation. Caffeine is also a potent non-specific inhibitor of various cellular phosphodiesterases (PDE) and its inhibitory effect can be reproduced by other PDE inhibitors, chemically related (theophylline) or not (papaverine). Various protocols designed to increase the concentration of either of the major intracellular cyclic nucleotides [adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP)] failed to reproduce the full extent of the caffeine inhibition: at maximal agonist concentration (1 μM ACh) increases of either cAMP or cGMP did not affect the Ca²⁺ signal, whereas at submaximal doses of agonist (0.1–0.3 μM ACh) they induced partial inhibition. Here we show that only the simultaneous increase of the cellular concentrations of both cyclic nucleotides (either simultaneous or sequential) are effective in mimicking the blocking effect of caffeine and other non-specific PDE inhibitors. These data indicate, thus, that, in addition to other independent intracellular effects, cAMP and cGMP can exert a co-ordinated inhibitory effect of the agonist-evoked Ca²⁺ signal in pancreatic acinar cells. Received: 25 March 1996/Accepted: 23 May 1996     

Reduced cholecystokinin receptor phosphorylation and restored signalling in protein kinase C down-regulated rat pancreatic acinar cells

 Receptor phosphorylation in response to agonist stimulation is a key regulatory principle in signal transduction. Previous work has suggested the concerted action of protein kinase C (PKC) and a staurosporine-insensitive receptor kinase in homologous phosphorylation of the cholecystokinin (CCK) receptor in freshly isolated rat pancreatic acinar cells [Gates, Ulrich, Miller (1993) Am J Physiol 264:G840–G847]. The present study shows that down-regulation of PKC by prolonged (2 h) treatment with 0.1 μM 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly reduced basal CCK receptor phosphorylation as well as that induced by TPA (0.1 μM) and cholecystokinin-(26-33)-peptide amide (CCK₈, 0.1 μM). The phosphorylation level reached was the same with both stimulants and equalled basal phosphorylation in untreated control cells. The absence of any CCK₈-stimulated phosphorylation reflecting the activity of a putative staurosporine-insensitive receptor kinase raises the intriguing possibility that a basal level of PKC-mediated receptor phosphorylation is required for the action of such a receptor kinase. Immunoblot analysis revealed that the decrease in receptor phosphorylation coincided with a marked reduction of PKC-α and, to a lesser extent, PKC-ɛ. In addition, TPA-induced inhibition of the increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) evoked by the high-affinity CCK receptor agonist JMV-180 was completely reversed. The time-course of recovery closely matched that of the reduction of PKC-α. Finally, digital imaging microscopy of individual PKC down-regulated cells revealed a marked increase in the duration of JMV-180-evoked oscillatory changes in [Ca²⁺]_i. Taken together, the present findings are in agreement with the idea that PKC-α-mediated receptor phosphorylation leads to a shortening of the duration of the [Ca²⁺]_i oscillations and eventually to inhibition of high-affinity Ca²⁺ signalling through the native CCK receptor in pancreatic acinar cells. Received: 15 September 1997 / Accepted: 2 October 1997     

Inhibition of suicidal erythrocyte death by nitric oxide

Nitric oxide (NO) is known to counteract apoptosis by S-nitrosylation of protein thiol groups. NO is generated and stored in erythrocytes, which may undergo eryptosis, a suicidal cell death similar to apoptosis of nucleated cells. Eryptosis is triggered by increased cytosolic Ca²⁺ activity and/or ceramide and characterized by cell shrinkage and phosphatidylserine exposure at the cell surface. The present study explored whether nitric oxide could interfere with the machinery underlying eryptosis. To this end, erythrocyte phosphatidylserine exposure (annexin V-binding) and cell volume (forward scatter) were determined by flow cytometry. The Ca²⁺ ionophore ionomycin (0.1 μM) increased cytosolic Ca²⁺ activity, triggered annexin binding, and decreased forward scatter. The annexin binding and decrease of forward scatter but not the increase of cytosolic Ca²⁺ activity were reversed by the NO-donor nitroprusside (1 μM) and papanonoate (100 μM). Higher concentrations of nitroprusside (0.1 and 1 mM) stimulated eryptosis. Glucose depletion, exposure to C₆-ceramide (3 μM), hypertonic (addition of 550 mM sucrose), and isotonic (replacement of Cl⁻ with gluconate) cell shrinkage all triggered annexin V binding, effects all reversed by nitroprusside (1 μM). Dibutyryl–cGMP (1 mM) blunted the ionomycin- but not the ceramide-induced annexin V binding. Ionomycin decreased protein nitrosylation and thioredoxin activity, effects reversed by the NO-donor papanonoate. Clearance of erythrocytes from circulating blood was significantly faster in eNOS knockout mice than in their wild-type littermates. In conclusion, nitric oxide participates in the regulation of erythrocyte survival, an effect partially mimicked by cGMP and paralleled by alterations of protein nitrosylation and thioredoxin activity. This study is dedicated to the memory of Rudi Busse, a unique scientist with outstanding sharp mind, strength, and dedication.     

Intracellular pH and calcium signalling in rat pancreatic acinar cells

 Intracellular free Ca²⁺ signals, which occur in many secretory cell types after the binding of some secretagogues to their membrane receptors, are due to Ca²⁺ mobilization from internal stores and Ca²⁺ influx from the extracellular space. There is also growing evidence for a modulatory role of intracellular pH in Ca²⁺ metabolism. In fact it has been proposed that Ca²⁺ stores in pancreatic acinar cells may be loaded by Ca²⁺/H⁺ exchange. The aim of this paper was to establish the effect of intracellular pH on Ca²⁺ signalling in pancreatic acinar cells. Application of the proton carrier nigericin impairs Ca²⁺ mobilization in response to cholecystokinin (CCK-8), and application of membrane-permeant bases or acids inhibits CCK-8-evoked intracellular Ca²⁺ oscillations. Both nigericin and a cell-permeant weak base release Ca²⁺ from internal stores. However, cytosolic acidification by removal of extracellular Na⁺ had no effect on the resting or stimulated cytosolic Ca²⁺ concentration. After depletion of Ca²⁺ stores by a maximal concentration of CCK-8, nigericin and ionomycin released a residual Ca²⁺ pool. Taken together, our results show that in pancreatic acinar cells Ca²⁺ signals require the existence of subcellular gradients of pH and indicate the presence of acidic pools of Ca²⁺. Received: 21 March 1997 / Received after revision: 14 May 1997 / Accepted: 15 May 1997     

The relationship between free cytosolic calcium and amylase release in rat pancreatic acini

We have studied the effects of various pancreatic secretagogues on free cytosolic calcium ([Ca2+]i) and amylase release in dispersed rat pancreatic acini, to determine the role of [Ca2+]i in stimulated enzyme secretion from the exocrine pancreas. Dispersed rat pancreatic acini were loaded with the new Ca2+-sensitive fluorescent indicator, fura-2. Resting [Ca2+]i was 110 +/- 2 nM (a mean +/- S.E.). Carbachol, caerulein, bombesin, and neuromedin B and C each caused a rapid increase in [Ca2+]i; maximal increases of 100 to 400-500 nM were reached within 20s following the secretagogue addition, and this was followed by a return to a lower sustained level within 2 min. When enzyme secretion from the acini was monitored as a function of time using a perifusion system, secretagogue-induced amylase release took a biphasic pattern consisting of an initial burst phase for a several minutes and a second sustained phase during stimulation. Although sustained amylase secretion occurred at near resting [Ca2+]i, the peak [Ca2+]i correlated with the amount of stimulated amylase release as well as with the initial release, during submaximal and maximal stimulation by these agents. At supramaximal concentrations of carbachol and caerulein, amylase release, but not the increase in [Ca2+]i, was attenuated. On the other hand, in response to supramaximal concentrations of bombesin, and neuromedin B and C, both the amount of amylase released and the peak [Ca2+]i were similar to those obtained in response to maximal concentrations. From a standpoint of time course analysis of enzyme secretion, both the first burst phase and the second sustained phase were inhibited during stimulation by 10(-3) M carbachol, compared with 10(-5) M carbachol, while supramaximal stimulation by neuromedin C caused a pattern of amylase release similar to that produced by maximal stimulation. These data suggest that in pancreatic acinar cells an increase in [Ca2+]i plays an important role in stimulus-secretion coupling; however, other factors may be indispensable in regulating enzyme secretion. Furthermore, it is suggested that there is a difference in the intracellular messenger system between carbachol and caerulein, and neurotransmitters belonging to the bombesin family, especially during supramaximal stimulations.     

Adenovirus-mediated gene expression in isolated rat pancreatic acini and individual pancreatic acinar cells

 In this study we have examined the feasibility of using replication-deficient recombinant adenoviral vectors to transfer and express genes in pancreatic acinar cellsin vitro. We infected primary cultures of both isolated pancreatic acini and individual acinar cells with a recombinant adenovirus containing the coding sequence for β-galactosidase. Our data demonstrate that recombinant adenoviruses readily infect pancreatic acinar cellsin vitro. Close to 100% infection and maximal β-galactosidase expression were obtained, when acini or acinar cells were infected with 5×10⁶ or 10⁶ plaque-forming units (pfu) of virus per millitre of acini or acinar cell suspension, respectively. Examination of the time-course of β-galactosidase expression showed that there was a lag of approximately 6 h before β-galactosidase levels increased. Thereafter β-galactosidase expression increased rapidly. By 20 h post-infection β-galactosidase activity had increased from undetectable levels to 2.5–3.0 units/mg of cellular protein. Acini/acinar cells maintained a robust secretory response after adenoviral infection. The cholecystokinin-octapeptide (CCK8) dose/response curves for amylase secretion for acini and acinar cells infected with 5×10⁵ and 1×10⁵ pfu/ml of virus, respectively, were biphasic, with maximal amylase secretion being stimulated by 1 nM CCK8. In addition, the dose/response curves were identical to those obtained from control, sham-infected, acini/acinar cells. Our findings indicate that replication-deficient recombinant adenoviral vectors will be excellent tools to transfer and express genes in isolated pancreatic acini or acinar cells. Received: 27 March 1998 / Received after remission: 6 May 1998 / Accepted: 7 May 1998     

Multisite regulation of insulin secretion by cAMP-increasing agonists: evidence that glucagon-like peptide 1 and glucagon act via distinct receptors

 The mechanisms by which glucagon-like peptide 1(7–36)amide (GLP-1[7–36]amide) potentiates insulin secretion were investigated by measurements of whole-cell K⁺ and Ca²⁺ currents, membrane potential, the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and exocytosis in mouse pancreatic B-cells. GLP-1(7–36)amide (10 nM) stimulated glucose-induced (10 mM) electrical activity in intact pancreatic islets. The effect was manifested as a 34% increase in the duration of the bursts of action potentials and a corresponding 28% shortening of the silent intervals. GLP-1(7–36)amide had no effect on the electrical activity at subthreshold glucose con- centrations (≤6.5 mM). In cultured B-cells, GLP-1(7–36)amide produced a decrease of the whole-cell ATP-sensitive K⁺ (K_ATP) conductance remaining at 5 mM glucose by ≈30%. This effect was associated with membrane depolarization and the initiation of electrical activity. GLP-1(7–36)amide produced a protein-kinase-A- (PKA-) and glucose-dependent fourfold potentiation of Ca²⁺-induced exocytosis whilst only increasing the Ca²⁺ current marginally. The stimulatory action of GLP-1(7–36)amide on exocytosis was mimicked by the pancreatic hormone glucagon and exendin-4, a GLP-1 receptor agonist. Whereas the stimulatory action of GLP-1(7–36)amide could be antagonized by exendin-(9–39), this peptide did not interfere with the ability of glucagon to stimulate exocytosis. We suggest that GLP-1(7–36)amide and glucagon stimulate insulin secretion by binding to distinct receptors. The GLP-1(7–36)amide-induced stimulation of electrical activity and Ca²⁺ influx can account for (maximally) a doubling of insulin secretion. The remainder of its stimulatory action results from a cAMP/PKA-dependent potentiation of Ca²⁺-dependent exocytosis exerted at a stage distal to the elevation of [Ca²⁺]_i. Received: 3 March 1997 / Received after revision and accepted: 23 May 1997     

Suppression of Ca2+ oscillations induced by cholecystokinin (CCK) and its analog OPE in rat pancreatic acinar cells by low-level protein kinase C activation without transition of the CCK receptor from a high- to low-affinity state

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 (InsP ₃) levels. In contrast, high concentrations of CCK elevate InsP ₃, 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°C did not affect OPE binding to acini, but abolished OPE-induced (at 1 M) [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 M). 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 M TPA inhibited OPE binding and caused a more generalized inhibition of OPE- and CCK-evoked Ca²⁺ signals. These data suggest that inhibitory effects of low-level PKC activation on agonist-evoked Ca²⁺ signalling are distinct from the effects of high-level PKC activation by 1 M TPA, and do not require the transition of the CCK receptor from a high-affinity to a low-affinity state.     

Effects of intracellular calcium on GABAA receptors in mouse cortical neurons

 Using the patch-clamp technique, we studied the effect of intracellular Ca²⁺ on Cl^– current gated by type A γ-aminobutyric acid receptors (GABA_A) in mouse cortical neurons. When the rapid Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) was in the pipette solution, the GABA-activated Cl^– current amplitude decreased over time to 49 ± 7% of control. In contrast, equimolar replacement of BAPTA with ethylenebis(oxonitrilo)tetraacetate (EGTA) caused a 60 ± 10% increase in GABA current. An increased intracellular Ca²⁺ concentration caused a transient augmentation of the GABA current. This effect of Ca²⁺ was concentration dependent (10 nM to 34 μM). Ca²⁺ increased the amplitude of the current by enhancing the maximal response to GABA rather than by changing the affinity of the receptor to GABA (EC₅₀ = 5 ± 0.4 μM vs. 7 ± 0.3 μM). Both calmodulin (CaM) and a CaM kinase II inhibitor (200 μM) blocked the potentiating effect of Ca²⁺ suggesting that it was mediated by activation of CaM kinase II. We found that regulation of GABA_A receptors by intracellular Ca²⁺ in cortical neurons has important physiological implications since the potentiating effect of increasing the intracellular Ca²⁺ on responses to GABA was mimicked by activating excitatory receptors with 100 μM N-methyl-D-aspartate (NMDA). These findings suggest that modulation of GABA_A receptor activity by glutamate may be brought about via changes in intracellular Ca²⁺. Received: 20 May 1997 / Received after revision: 12 August 1997 / Accepted: 1 September 1997     

Calcium-contraction relationship in rat mesenteric arterial smooth muscle. Effects of exogenous and neurogenic noradrenaline

 We evaluated the relationship between intracellular calcium concentration ([Ca²⁺]_i) and vasoconstriction during the presence of exogenous noradrenaline (NA) and sympathetic nerve stimulation. An imaging technique was used to determine calcium/tension relationships in isolated rat mesenteric resistance arteries that had been mounted for recording of isometric tension development and loaded with Fura-2/AM. Experiments were performed after depletion of vasodilator neuropeptides and in the continuous presence of 1 μM propranolol, 3 μM indomethacin, and 30 μM nitro-l-arginine. NA (10 μM) was shown first to induce a further increase in tension, but not [Ca²⁺]_i, during the contraction induced by 125 mM K⁺. Subsequently, calcium/tension relationships were determined during stimulation with graded increases in extracellular [K⁺] (5.9–125 mM K⁺), cumulative administration of NA (0.2–10 μM) and electrical field stimulation of perivascular nerves (EFS, 1–16 Hz). A basal calcium/tension relationship without the calcium-sensitizing property of NA was constructed using a cumulative concentration/response curve of 5.9–125 mM K⁺ in arteries after prior exposure to the irreversible α-adrenoceptor antagonist phenoxybenzamine (POB). K⁺ series before and during α-blockade were also studied using the combination of the α₁-antagonist prazosin and α₂-antagonist yohimbine yielding comparable results as with POB. Calcium/tension curves obtained in the presence of NA, K⁺ and during EFS all were shifted to the left compared with the basal condition and all showed a similar slope indicating that neurogenically released NA is equally capable of inducing calcium sensitization in smooth muscle of mesenteric resistance arteries as exogenously applied NA. In the presence of exogenous and endogenous NA we not only observed an elevated contractile response for a given increase in [Ca²⁺]_i, but also an attenuated rise in [Ca²⁺]_i for a given intensity of stimulation. This suggests that the agonist-induced calcium-sensitization is accompanied by a reduction of the rise in [Ca²⁺]_i. Received: 24 October 1997 / Received after revision: 26 January 1998 / Accepted: 26 February 1998     

Q-type Ca2+ channels are located closer to secretory sites than L-type channels: functional evidence in chromaffin cells

 This study uses a new strategy to investigate the hypothesis that, of the various Ca²⁺ channels expressed by a neurosecretory cell, a given channel subtype is coupled more tightly to the exocytotic apparatus than others. The approach is based on the prediction that the degree of inhibition of the secretory response by various Ca²⁺ channel blockers will differ at low (0.5 mM) and high (5 mM) extracellular Ca²⁺ concentrations ([Ca²⁺]_o). So, at low [Ca²⁺]_o the K⁺-evoked catecholamine release from superfused bovine chromaffin cells was depressed 60–70% by 2 μM ω-agatoxin IVA (P/Q-type Ca²⁺ channel blockade), by 3 μM ω-conotoxin MVIIC (N/P/Q-type Ca²⁺ channel blockade), or by 3 μM lubeluzole (N/P/Q-type Ca²⁺ channel blockade); in high [Ca²⁺]_o these blockers inhibited the responses by only 20–35%. At 1–3 μM ω-conotoxin GVIA (N-type Ca²⁺ channel blockade) or 3 μM furnidipine (L-type Ca²⁺ channel blockade), secretion was inhibited by 30 and 50%, respectively; such inhibitory effects were similar in low or high [Ca²⁺]o. Combined furnidipine plus ω-conotoxin MVIIC, ω-agatoxin IVA or ω-conotoxin GVIA exhibited additive blocking effects at both Ca²⁺ concentrations. The results suggest that Q-type Ca²⁺ channels are coupled more tightly to exocytotic active sites, as compared to L-type channels. This hypothesis if founded in the fact that external Ca²⁺ that enters the cell through a Ca²⁺ channel located near to chromaffin vesicles will saturate the K⁺ secretory response at both [Ca²⁺]_o, i.e. 0.5 mM and 5 mM. In contrast, Ca²⁺ ions entering through more distant channels will be sequestered by intracellular buffers and, thus, will not saturate the secretory machinery at lower [Ca²⁺]_o. Received: 23 September 1997 / Received after revision: 29 October 1997 / Accepted: 30 October 1997     

Ionic and osmotic dependence of secretion from permeabilised acini of the rat pancreas

Many types of secretory granule have been observed to swelll as a result of cell stimulation implying a degree of osmotic control, although the regulation of granule fusion with the apical plasma membrane is not clearly understood. In the present study we have investigated the ionic and osmotic dependency of basal and stimulated³H-protein release from rat pancreatic acini, permeabilised by either digitonin or high voltage electric discharge. Acini were stimulated with either cholecystokinin-pancreozymin octapeptide (CCK-8), carbachol (CCh), or with phorbol ester (TPA) plus cAMP. Stimulated secretion was significantly reduced when 130 mmol/l Cl^– in the buffer was replaced by I^–, NO ₃ ^– , SCN^– or cyclamate^–. Secretion in Cl^– buffers was inhibited by the anion transport inhibitor 4,4-diisothiocyanatostibene-2,2-disulfonic acid (DIDS), by 40% of the control response. Neither Na⁺ nor N-methyl-d-glucamine^– could replace K⁺ in the buffer. Ba²⁺ and quinine, which block K⁺ conductance pathways, inhibited stimulated secretion by 50%. Finally, stimulated secretion from leaky cells was nearly abolished by doubling buffer osmolarity. The data suggest that when the cell is stimulated, a Cl^– and a K⁺ permeability appear in the zymogen granule membrane and the ions enter down their electrochemical gradients. The increased intragranular osmolarity results in granular swelling which is intimately associated with secretion.Parts of this study have been previously presented at the XIXth Meeting of the European Pancreatic Club (1987) and at the 65th Meeting of the Deutsche Physiologische Gesellschaft (1988)     

cGMP reduces the sarcoplasmic reticulum Ca<Superscript>2+</Superscript> loading in airway smooth muscle cells: a putative mechanism in the regulation of Ca<Superscript>2+</Superscript> by cGMP

Ca²⁺ and cGMP have opposite roles in many physiological processes likely due to a complex negative feedback regulation between them. Examples of opposite functions induced by Ca²⁺ and cGMP are smooth muscle contraction and relaxation, respectively. A main Ca²⁺ storage involved in contraction is sarcoplasmic reticulum (SR); nevertheless, the role of cGMP in the regulation of SR-Ca²⁺ has not been completely understood. To evaluate this role, intracellular Ca²⁺ concentration ([Ca²⁺]i) was determinated by a ratiometric method in isolated myocytes from bovine trachea incubated with Fura-2/AM. The release of Ca²⁺ from SR induced by caffeine was transient, whereas caffeine withdrawal was followed by a [Ca²⁺]i undershoot. Caffeine-induced Ca²⁺ transient peak and [Ca²⁺]i undershoot after caffeine were reproducible in the same cell. Dibutyryl cGMP (db-cGMP) blocked the [Ca²⁺]i undershoot and reduced the subsequent caffeine peak (SR-Ca²⁺ loading). Both, the opening of SR channels with ryanodine (10 μM) and the blockade of SR-Ca²⁺ ATPase with cyclopiazonic acid inhibited the [Ca²⁺]i undershoot as well as the SR-Ca²⁺ loading. The addition of db-cGMP to ryanodine (10 μM) incubated cells partially restored the SR-Ca²⁺ loading. Cyclic GMP enhanced [Ca²⁺]i undershoot induced by the blockade of ryanodine channels with 50 μM ryanodine. In conclusion, the reduction of SR-Ca²⁺ content in airway smooth muscle induced by cGMP can be explained by the combination of SR-Ca²⁺ loading and the simultaneous release of SR-Ca²⁺. The reduction of SR-Ca²⁺ content induced by cGMP might be a putative mechanism limiting releasable Ca²⁺ in response to a particular stimulus.     

Blockade of nitric oxide formation inhibits the stimulation of the renin system by a low salt intake

This study aimed to investigate the possible involvement of endothelial autacoids such as nitric oxide or prostaglandins in the well-known stimulatory effect of a low salt intake on renin secretion and renin gene expression in the kidney. To this end, plasma renin activity (PRA) and kidney renin mRNA levels were determined in male Spargue-Dawley rats fed either a normal (0.6% w/w) or a low (0.03%) NaCl diet for 10 days. To inhibit nitric oxide formation, the animals received L-nitro-argininemethylester (L-NAME, 40 mg/ kg twice a day), to inhibit prostaglandin formation the animals received meclofenamate (8 mg/kg twice a day) during the last 2 days. In animals fed a normal salt diet, L-NAME decreased PRA from 6.5 to 4.9 ng angiotensin I⋅h⁻¹⋅ml⁻¹ and decreased renin mRNA levels by about 15%. Meclofenamate did not change PRA or renin mRNA in animals fed on normal salt diet. In vehicle-treated animals fed a low salt diet, PRA increased from 6.5 to 20.2 ng ANGI⋅h⁻¹⋅ml⁻¹ and renin mRNA levels increased by 100%. Meclofenamate treatment did not alter these changes of PRA and renin mRNA during the intake of a low salt diet. In animals treated with L-NAME, PRA increased to only 7.2 ng ANGI⋅h⁻¹⋅ml⁻¹ and renin mRNA increased by 20%. These findings indicate that inhibition of nitric oxide formation but not of prostaglandin formation substantially attenuates the stimulatory effect of a low salt intake on the renin system, suggesting that nitric oxide is required for this process. Received: 21 September 1995/Received after revision: 18 January 1996/Accepted: 8 February 1996     

Early effects of denervation on sarcoplasmic reticulum properties of slow-twitch rat muscle fibres

 The Ca²⁺ release activity of the sarcoplasmic reticulum (SR) in chemically skinned single slow-twitch fibres from control, 2-day and 7-day denervated rat soleus muscle was studied. Histochemical fibre type composition of the whole muscle, electrophysiological properties and the Ca²⁺ sensitivity of tension development by single muscle fibres were also studied. All the data were correlated with contractile properties of the in vitro muscle. In the 2-day denervated muscle the SR Ca²⁺ capacity and the rate of Ca²⁺ uptake decreased from the control values of 0.384 ± 0.030 μmol (mg fibre protein)^–1 and 19.8 ± 1.9 nmol min^–1 (mg fibre protein)^–1, respectively, to 0.210 ± 0.016 μmol (mg fibre protein)^–1 and 13.5 ± 0.9 nmol min^–1 (mg fibre protein)^–1; the calculated amount of Ca²⁺ released upon stimulation by caffeine decreased from the control value of 0.148 to 0.078 μmol (mg fibre protein)^–1. In the 7-day denervated muscle, the SR Ca²⁺ capacity and the rate of Ca²⁺ uptake increased to 0.517 ± 0.06 μmol (mg fibre protein)^–1 and 21.6 ± 2.3 nmol min^–1 (mg fibre protein)^–1, respectively; the calculated amount of Ca²⁺ released increased to 0.217 μmol (mg fibre protein)^–1. Both contraction time and tension of the isometric twitch decreased in 2-day denervated and increased in 7-day denervated muscles. Electrophysiological and histochemical changes, as well as changes in the Ca²⁺ sensitivity of the muscle fibres did not show any apparent correlation with mechanical changes. It is therefore concluded that the SR plays a prominent role in the early changes of contraction time and tension following denervation. Received: 15 October 1996 / Received after revision: 28 March 1997 / Accepted: 8 April 1997     

μ and δ opioid receptor activation inhibits ω-conotoxin-sensitive calcium channels in a voltage- and time-dependent mode in the human neuroblastoma cell line SH-SY5Y

 Ca²⁺ channel modulation by the μ opioid agonist [d-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin (DAGO) and the δ opiate agonists [d-Pen², d-Pen⁵]-enkephalin (DPDPE) and [d-Ala², d-Leu⁵]-enkephalin (DADLE) in cultured human neuroblastoma SH-SY5Y cells was investigated using the whole-cell variant of the patch-clamp technique. In SH-SY5Y cells, differentiated in vitro with retinoic acid, all agonists reversibly decreased high-voltage-activated, ω-conotoxin-sensitive Ba²⁺ currents in a concentration-dependent way. Inhibition was maximal with a 1 μM concentration of opiate agonists (76% with DAGO and 63% with δ agonists, when measured at 0 mV) and was characterized by a clear slow down of Ba²⁺ current activation at low test potentials. Both inhibition and slow down of activation were attenuated at more positive potentials, and could be partially relieved by strong conditioning depolarizations. Current suppression operated by both μ and δ agonists was prevented by pre-treatment of the cells with pertussis toxin. No sign of additivity was observed when δ agonists were applied to cells that were maximally activated by DAGO, suggesting that a common mechanism, involving the same type of modulating molecule, is responsible for Ca²⁺ channel inhibition promoted by activation of μ and δ opioid receptors in SH-SY5Y cells. Received: 10 October 1996 / Received after revision and accepted: 18 November 1996     

Endothelium derived relaxing factor is involved in the pressure control of renin gene expression in the kidney

To study the influence of endothelium derived relaxing factor/nitric oxide (EDNO) on renin gene expression, the effects of a 2-day treatment with the NO-synthase inhibitor nitro-L-arginine-methylester (L-NAME, 40 mg/kg twice a day) on plasma renin activity (PRA) and renal and adrenal renin m-RNA levels were examined in conscious rats with and without unilateral renal clips (0.2 mm). In sham-clipped animalsL-NAME led to a decrease of PRA from 7.5 to 2.5 ng angiotensin I (ANGI) · h^–1 · ml^–1 and to a 35% decrease of renal renin m-RNA levels. Unilateral renal artery clipping increased PRA to 35 and to 13 ng ANGI · h^–1 · ml^–1 in vehicle and inL-NAME-treated rats, respectively. In the clipped kidneys renin m-RNA levels increased to 450% of control values in vehicle-treated animals and to 220% of control values inL-NAME-treated animals. In the contralaterals as opposed to clipped kidneys, renin m-RNA levels decreased to 16% and 50% of the control values in vehicle- and inL-NAME-treated animals, respectively. In the adrenal glands renin m-RNA levels were not significantly changed either by clipping of one renal artery or by treatment of animals withL-NAME. The NO-donor sodium nitroprusside (100 M) was found to increase renin secretion and renin m-RNA levels in primary cultures of renal juxtaglomerular cells. These findings suggest that EDNO is involved in the control of the renin gene by the renal perfusion pressure.     

A calcium-dependent chloride current in insulin-secreting βTC-3 cells

 Ca²⁺-dependent conductances have been hypothesized to play a role in the bursting pattern of electrical activity of insulin-secreting β cells in response to high plasma glucose. A Maxi K⁺ channel has received the most attention, while a low-conductance Ca²⁺-activated K⁺ current has also been identified. We used an increasingly popular β cell model system, the βTC-3 cell line, and the perforated-patch technique to describe the properties of a novel Ca²⁺-dependent Cl^–current [I _Cl(Ca)] in insulin-secreting pancreatic β cells. The reported I_Cl(Ca) could be activated under physiological Ca²⁺ concentrations and is the first of its kind to be described in pancreatic insulin-secreting cells. We found that long depolarizing steps above –20 mV elicited an outward current which showed slow inward relaxation upon repolarization to negative membrane potentials. Both the outward currents and the inward tails showed dependence on Ca²⁺ influx: their current/voltage (I/V) relations followed that of the ”L-like” Ca²⁺ current (I _Ca) present in these cells; they were blocked completely by the removal of external Ca²⁺ or application of Cd²⁺ at concentrations sufficient for complete block of I _Ca; and their magnitude increased with the depolarizing step duration. Moreover, the inward tail decayed monoexponentially with a time constant which at voltages negative to activation of I _Ca showed a weak linear voltage dependence, while at voltages positive to activation of I _Ca it followed the voltage dependence of I _Ca. This Ca²⁺-dependent current reversed at –21.5 mV and when the external Cl^–concentration was reduced from 159 mM to 62 mM the reversal potential shifted by ≈+20 mV as predicted by the Nernst relation for a Cl^–-selective current. Cl^–channel blockers such as DIDS (100 μM) and niflumic acid (100 μM) blocked this current. We concluded that this current was a Ca²⁺-dependent Cl^–current [I _Cl(Ca)]. From substitution of the external Cl^–with various monovalent anions and from the reversal potentials we obtained the following permeability sequence for I _Cl(Ca): I ^–>NO₃ ^–>Br^–>Cl^–>Acetate^–. Received: 10 October 1996 / Received after revision and accepted: 19 December 1996     

Activation of non-selective cation conductance by carbachol in freshly isolated bovine ciliary muscle cells

 In smooth muscle cells freshly isolated from the bovine ciliary body, effects of carbachol (CCh) on the membrane potential and current were examined by the whole-cell clamp method. The resting membrane potential of the muscle cells used was –60 ± 1 mV (n=111). Extracellular application of CCh (2 μM) depolarized the cells to –15 ± 5 mV (n=50) with an apparent increase in membrane conductance. Under voltage-clamp conditions, CCh (2 μM) evoked an inward current which exhibited inward-going rectification and reversed the polarity at about 0 mV. Removal of Na⁺ from the external solution caused a reduction of the amplitude of the current and a shift of the reversal potential to the negative direction. CCh was able to elicit an inward current even under a condition where Ca²⁺ was the only cation producing an inwardly directed electrochemical gradient. The current was not affected by verapamil or by tetrodotoxin. The CCh-induced current was inhibited by antimuscarinic agents with the affinity sequence: atropine ≈4–DAMP >> pirenzepine > AF-DX116, indicating that the response is mediated by a muscarinic cholinoceptor that belongs to the M₃-subtype. Unlike the non-selective cation channel current in intestinal smooth muscles, which is activated by elevation of the intracellular Ca²⁺ concentration ([Ca²⁺]_i), the current of the ciliary muscle was inactivated when the [Ca²⁺]_i was increased. The conductance, which admits Ca²⁺, may serve as a pathway for Ca²⁺ entry required for contraction. Received: 2 December 1996 / Received after revision: 7 January 1997 / Accepted: 8 January 1997