PGE2-induced inhibition of AVP-dependent cAMP accumulation in the OMCD of the rat kidney is cumulative with respect to the effects of α2-adrenergic and A1-adenosine agonists,insensitive to pertussis toxin and dependent on extracellular calcium

The accumulation of cyclic adenosine 3,5-phosphate (cAMP) elicited by antidiuretic hormone (arginine vasopressin, AVP) in the medullary collecting tubule (OMCD) microdissected from the rat kidney is inhibited by different factors: the A₁ agonist of adenosine (-)-N ⁶-(R-phenylisopropyl) adenosine (PIA), an ₂-adrenergic agonist clonidine (CLO), and prostaglandin E₂ (PGE₂). The negative regulation elicited by PGE₂ was further characterized by measuring summation of inhibition with other inhibitors, by testing the effect of pertussis toxin and by studying the part played by extracellular calcium. Inhibitors were used at concentrations inducing maximum effects. The simultaneous addition of 0.3 M PGE₂ with either 0.1 M PIA or 1 M CLO led to an inhibition of the response to AVP (80.0±3.5%, SEM, N=7 and 92.6±0.8%, N=5, respectively) greater than those elicited by each agent alone. In contrast, PIA and CLO added together induced an inhibition similar to that due to CLO alone. The action of PGE₂ in combination with either PIA or CLO corresponded to a partial summation fitting with the values calculated by assuming a cumulative inhibition. Preincubation of OMCD samples with pertussis toxin (100 ng/ml or 1 g/ml) relieved the inhibitory effects of CLO and PIA but did not affect the action of PGE₂. PGE₂-induced inhibition was prevented in a calcium-free medium [0 Ca²⁺+0.1 mM [ethylene-bis (oxyethylenenitrilo)] tetraacetate (EGTA)]: values were 67.0 ±2.1% and 5.8±8.7% (± SEM) in 2 mM Ca²⁺ and 0 Ca²⁺ medium, respectively, N=7. When applied to Fura-2-loaded OMCD, 0.3 M PGE₂ increased intracellular calcium concentration ([Ca²⁺]_i) with a peak phase (in 2 mM or 0 Ca²⁺ medium) followed by a plateau phase (observed only in 2 mM Ca²⁺ medium). It is concluded that: (1) in the rat OMCD, PGE₂, PIA and CLO act on the same AVP-sensitive cell, (2) PGE₂ induces a cumulative inhibition on the cAMP level when combined with other inhibitors by a mechanism insensitive to pertussis toxin, (3) the presence of extracellular calcium is a prerequisite condition to observe PGE₂-induced inhibition, and (4) the inhibition by PGE₂ might be linked to its capacity of increasing [Ca²⁺]_i.     

Effect of calcitonin on the regulation of intracellular pH in primary cultures of rabbit early distal tubule

To examine the intracellular pH (pH_i) regulation in primary cultures of rabbit distal convoluted tubules (DCTb) we used the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM) and a video-microscopy technique. DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The culture epithelia were grown on semi-transparent permeable supports. Before pH_i measurement, DCTb primary cultures were maintained for 48–96 h in growth-factor-free medium to obtain quiescent cells. We had previously shown that two mechanisms are involved in the regulation of intracellular pH: a basolateral Na⁺/H⁺ exchanger and an apical Cl^–/HCO ₃ ^– exchanger [1]. The pH_i of DCTb cells was significantly decreased by the addition of 60 nM human calcitonin (from 7.30±0.04 to 7.08±0.04). This response to calcitonin was dose-dependent and mimicked by both forskolin and permeant cyclic AMP derivatives. An initial acidification (of 0.25 pH unit in 7–8 min) was observed after the addition of basolateral amiloride (1 mM). The persistence of the effect induced by human calcitonin in these conditions, suggests that the Na⁺/H⁺ exchanger is not involved in the response. However, the acidification response was blocked in both the absence of chloride at the apical side and by the apical addition of 0.1 mM 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS). These experiments suggest that the target for the human calcitonin effect on pH_i is the Cl^–/HCO ₃ ^– exchanger. This study confirms the importance of this transporter in pH_i regulation within the physiological pH_i range and the influence of calcitonin in the regulation of DCTb cell function.     

The activation of protein kinase C prevents PGE2-induced inhibition of AVP-dependent cAMP accumulation in the rat outer medullary collecting tubule

Previous studies have demonstrated that prostaglandin E₂ (PGE₂) inhibits arginine vasopressin-(AVP)dependent adenosine 3,5-cyclic monophosphate (cAMP) accumulation in microdissected rat outer medullary collecting tubules (OMCD), by a mechanism unrelated to the inhibition of cAMP synthesis. The potential role of the activation of protein kinase C (PKC) to explain the negative regulation elicited by PGE₂ was investigated in this study. Single OMCD samples were pre-incubated (10 min, 30°C) in the presence or absence of either activators of PKC, phorbol 12-myristate 13-acetate (PMA), 1-oleoyl-2-acetyl-glycerol (OAG), dioctanoylglycerol (DOG) or an inhibitor of PKC, staurosporine (SSP). These compounds were present also with the agonists tested during the incubation period (4 min, 35°C). In contrast to PGE₂, activators of PKC did not decrease AVP-dependent cAMP accumulation (mean ±SEM): 1nM AVP=47.1±6.8 fmol · mm^–1· 4 min^–1; AVP + 0.3 M PGE₂=20.1±2.7, P<0.01 versus AVP; AVP + 10 nM PMA=42.0±4.7, NS versus AVP; AVP + 50 g/ml OAG=44.1±4.8. NS versus AVP, N= 5 experiments. However, 10 nM PMA prevented PGE₂-induced inhibition: AVP + PGE₂= 44.2±3.5% of the response to AVP and 90.3±3.2% without and with PMA respectively, N= 16. Similar results were obtained with either 50 g/ml OAG or 25 g/ ml DOG (AVP + PGE₂ + OAG=92.9±6.6% of the response to AVP, N= 8; AVP + PGE₂ + DOG=94.1 ±5.3%, N= 7). OAG, DOG, PMA or PMA + PGE₂ had no intrinsic agonist activity in the rat OMCD and the addition of an inactive phorbol ester did not prevent PGE₂-induced inhibition. SSP, 50 nM or 0.1 M, did not affect the inhibition due to PGE₂ but abolished the reversion by PMA of PGE₂-induced inhibition. A similar regulation was observed on forskolin-(FK)dependent cAMP accumulation: 5 M FK + 0.3 M PGE₂= 37.7±6.2% of the response to FK; FK + PGE₂ + 10 nM PMA=89.5±6.7%; FK + PGE₂ + PMA + 0.1 M SSP=43.1±7.9%, N= 4. The inhibition induced by an ₂-adrenergic agonist, clonidine 1 M, was not blocked by the activation of PKC. In fura-2-loaded OMCD samples, 10nM PMA decreased by 63.3±5.0% and by 57.2±7.1% the peak and plateau phases, respectively, of the increase in intracellular calcium concentration ([Ca²⁺]_i) obtained with PGE₂ when compared to control responses in the same tubules (n=12) and did not affect the increase in [Ca²⁺]_i induced by 0.1 mM carbachol. It is concluded that: (1) in the rat OMCD the activation of PKC by PMA or analogues of diacylglycerol did not reproduce PGE₂-induced inhibition of AVP- or FK-dependent cAMP accumulation, but prevented specifically this inhibitory action; and (2) this reversion might be the consequence of the effect of PKC activation which impaired the rises in [Ca²⁺]_i induced by PGE₂.     

The effect of secretagogues on ion conductances of in vitro perfused,isolated rabbit colonic crypts

Several secretagogues were used in this study, including those which enhance intracellular cyclic adenosine monophosphate (cAMP) production, as well as others which elevate intracellular Ca²⁺ activity and are known to increase Cl^– secretion in the intact colon and in colonic carcinoma cell lines. They were examined with respect to their effects on electrophysiological properties in isolated rabbit distal colonic crypts. Crypts were dissected manually and perfused in vitro. Transepithelial voltage (V _te), transepithelial resistance (R _te), membrane voltage across the basolateral membrane (V _bl), and fractional basolateral membrane resistance (FR _bl), were estimated. Basolateral prostaglandin E₂ (PGE₂, 0.1 mol/l), vasoactive intestinal peptide (VIP, 1 nmol/l) and adenosine (0.1 mmol/l) induced an initial depolarisation and a secondary partial repolarisation of (V _bl). In the case of adenosine, the initial depolarization of (V _bl) was by 31±2 mV (n=47).R _te fell significantly from 16.4±3.6 to 14.2±3.7 ·cm² (n= 6), andFR _blincreased significantly from 0.11±0.02 to 0.51±0.10 (n=6). In the second phase the repolarisation of (V _bl) amounted 11±2 mV (n=47) and a steadystate (V _bl) of –51±2 mV (n=47) was reached.R _te fell further and significantly to a steady-state value of 12.4±3.8 ·cm² (n=6) andFR _bl fell significantly to 0.42±0.13 (n=6). In 30% of the experiments, a transient hyperpolarisation of (V _bl) by 8±2 mV (n=14) was seen during wash out of adenosine. In the presence of adenosine, but not under control conditions, lowering of luminal Cl^– concentration from 120 to 32 mmol/l depolarised (V _bl) significantly by 8±1 mV (n=9). Basolateral ATP and ADP (0.1 mmol/l) led to a short initial depolarisation followed by a sustained and significant hyperpolarisation by 6±2 mV (n=27) and 5±4 mV (n=8), respectively. Carbachol (CCH) hyperpolarised (V _bl) in a concentration-dependent manner. At 100 mol/l (bath) the hyperpolarisation was by 14±2 mV (n=11) andFR _bl fell slightly. Neurotensin (10 nmol/l), isoproterenol (10 mol/l) and uridine 5-triphosphate (UTP, 0.1 mmol/l) had no effect. It is concluded that PGE₂, VIP and adenosine upregulate sequentially a luminal Cl^– conductance and a basolateral K⁺ conductance by increasing intracellular cAMP concentration. Ca²⁺ mobilising hormones such as ATP, ADP, and CCH increase the basolateral K⁺ conductance, while the effect on luminal Cl^– conductance appears to be very limited.     

Localization and properties of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase activity in the rat kidney

Localization of NAD⁺-dependent (type I) 15-hydroxyprostaglandin dehydrogenase (15PGDH) in the rat kidney was examined using an ultramicro assay of the enzyme activity based on the enzymatic cycling method. The enzyme activities during first 3 weeks of age were 30- to 40-fold higher than the adult and rapidly decreased by 4th week. 15PGDH activities measured with either PGE₂ or PGF₂ as a substrate were five times higher in slices from midcortical or juxtamedullary layers than in slices from the superficial cortex of 3 week-old rat kidney. Little activity was found in inner medulla and papilla. When the enzyme activity was assayed using isolated nephron segments dissected from collagenase treated slices of 3 week-old rat kidneys, the activity was localized only in the proximal convoluted and straight tubules with either PGs (PGE₂: 1.75±0.25 in PCT, 7.70±1.19 in PST, and PGF₂: 1.63±0.39, 6.18±1.52 pmoles NADH/mm/40 min). The kinetic analysis for renal 15PGDH of 3 week-old rats revealed thatK _m for PGE₂ (8.4 M) was lower than that for PGF₂ (22.6 M) with constant NAD⁺, whileV _max for both was similar. In contrast, bothK _m andV _max for NAD⁺ were identical with either PGs. These data suggest that the rate-limiting factor of type I 15PGDH is the concentration of prostaglandins in the kidney rather than the concentration of NAD⁺. It is thought that the enzyme may be physiologically significant in inactivating the prostaglandins reaching the proxial tubule in order to ensure that the classical prostaglandins (PGE₂ and PGF₂) produced in the medullary interstitium and collecting tubules can effectively regulate the distal nephron function.     

A new class of inhibitors of cAMP-mediated Cl− secretion in rabbit colon,acting by the reduction of cAMP-activated K+ conductance

Previously we have shown that arylamino-benzoates like 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which are very potent inhibitors of NaCl absorption in the thick ascending limb of the loop of Henle, are only poor inhibitors of the cAMP-mediated secretion of NaCl in rat colon. This has prompted our search for more potent inhibitors of NaCl secretion in the latter system. The chromanole compound 293 B inhibited the equivalent short-circuit current (I _sc) induced by prostaglandin E₂ (n=7), vasoactive intestinal polypeptide (VIP,n=5), adenosine (n=3), cholera toxin (n=4) and cAMP (n=6), but not by ionomycin (n=5) in distal rabbit colon half maximally (IC₅₀) at 2 mol/l from the mucosal and at 0.7 mol/l from the serosal side. The inhibition was reversible and paralleled by a significant increase in transepithelial membrane resistance [e.g. in the VIP series from 116±16 ·cm² to 136±21 ·cm² (n=5)]. A total of 25 derivatives of 293 B were examined and structure activity relations were obtained. It was shown that the racemate 293 B was the most potent compound with-in this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 mol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 mol/l 293 B had no effect on the membrane voltage across the basolateral membrane (V _bl) in non-stimulated crypt cells: –69±3 mV versus –67±3 mV (n=10), whilst in the same cells 1 mmol/l Ba²⁺ depolarised (V _bl) significantly. However, 293 B depolarised (V _bl) significantly in the presence of 1 mol/l forskolin: –45±4mV versus –39±5 mV (n=7). Similar results were obtained with 0.1 mmol/l adenosine. 293 B depolarised (V _bl) from –40±5 mV to –30±4 mV (n=19). This was paralleled by an increase in the fractional resistance of the basolateral membrane. VIP had a comparable effect. The hyperpolarisation induced by 0.1 mmol ATP was not influenced by 10 mol/l 293 B: –75±6 mV versus –75±6 mV (n=6). Also 293 B had no effect on basal K⁺ conductance (n=4). Hence, we conclude that 293 B inhibits the K⁺ conductance induced by cAMP. This conductance is apparently relevant for Cl^– secretion and the basal K⁺ conductance is insufficient to support secretion.     

Activation of nonselective cation channels in the basolateral membrane of rat distal colon crypt cells by prostaglandin E2

Ion channels in the basolateral membrane of colonic crypts were investigated with the patch-clamp technique during stimulation of secretion. Intact crypts were isolated from rat distal colon and the cell potential was recorded by addition of nystatin to the pipette solution. The cell resting potential in the base of the crypt was –74±1 mV (n=90). Addition of 100 M carbachol to the bath resulted in a transient hyperpolarization by 9 mV, which was probably due to the opening of basolateral K⁺ channels. In contrast, application of prostaglandin E₂ (PGE₂, 1 nM–1 M) caused a dose-dependent depolarization in the base of the crypt. With 1 M PGE₂ cells depolarized from -74±1 to –27±2 mV (n=26). Cell potential recordings in the midcrypt showed only a slight and transient depolarization after application of PGE₂, whereas cells close to the surface of the crypt had no response. In the base of the crypt the PGE₂-induced depolarization could be completely inhibited by addition of 50 M flufenamic acid, a known blocker of nonselective cation channels. After substitution of all monovalent cations by N-methyl-D-glucamine in the bath, PGE₂ had no significant effect on the cell potential. Cell-attached experiments with no nystatin in the patch pipette revealed the activation of ion channels in the basolateral membrane after application of PGE₂. After excision of the membrane patch, these channels could be identified as nonselective cation channels. Experiments involving substitution of the bath solution showed that the channel is impermeable for Cl^– and scarcely permeable for Ca²⁺ ions. The permeability sequence for monovalent cations, as calculated from reversal potentials, is NH ₄ ⁺ >Na⁺=K⁺>Rb⁺=Li⁺TRIS⁺=NMDG⁺. Single channels are completely inhibited by flufenamic acid (50 M), mefenamic acid (200 M), as well as by 3, 5-dichlorodiphenylamine-2-carboxylate. In conclusion, PGE₂ activates nonselective cation channels in the basolateral membrane of cells in the base of colonic crypts. It is suggested that this mechanism initiates the secretion of K⁺ ions. Na⁺ influx through the nonselective cation channel will stimulate the Na⁺/K⁺ pump and active uptake of K⁺ at the basolateral side. K⁺ can leave the cell at the luminal side through K⁺-selective channels.     

Synthesis and release of platelet-activating factor and eicosanoids in human endothelial cells induced by different agonists

Production of platelet-activating factor (PAF) and eicosanoids by human umbilical vein endothelial cells (HUVEC) after stimulation with different agonists has been studied. Significant amounts of PAF were measured in the cellular fraction after treatment with thrombin (2 NIHu/ml), calcium ionophore A23187 (2 M) and histamine (100 M) (110.3±14.3, 80.7±19.2 and 119.2±22.4 pg/10⁵ cells, respectively). Only thrombin caused a partial release of PAF into the supernatant. IL-1 (0.1 nM), TNF (1 nM), arachidonic acid (10 M) and endothelin (0.1 M) were not able to induce any PAF synthesis. High levels of 6-keto-PGF₁ were found after stimulation with thrombin and calcium ionophore A23187 (8641±2575 and 6715±3340 pg/10⁵ cells, respectively). Cytokines IL-1 and TNF were also able to stimulate PGI₂ synthesis, although to a lesser extent. PGE₂ production increased after treatment with thrombin and calcium ionophore A23187 three- and two-fold, respectively. Our results confirm that stimulated HUVEC are able to synthesize PAF and eicosanoids simultaneously, the relative amounts depending upon the agonist used. None of the agonists studied showed any significant effect on 15-HETE production.     

Evidence that arachidonic acid derived from neutrophils and prostaglandin E<Subscript>2</Subscript> are associated with the induction of acute lung inflammation by lipopolysaccharide of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objective: The involvement of arachidonic acid (AA) and PGE₂ during the E. coli lipopolysaccharide (LPS)-induced acute lung injury was investigated.Material: Adult male Wistar rats were used. For in vitro studies, rat neutrophils, bronchoalveolar lavage (BAL) fluid, and lug vascular endothelium were used, as described below.Treatment: Rats were given an intratracheal injection of LPS (750 g).Methods: Total and differential cell counts in BAL fluid; enzyme-linked immunoassay (ELISA) analyses of TNF-, IL-1, LTB₄ and PGE₂ in BAL, and immunohistochemical detection of ICAM-1 on lung vascular endothelium were performed six h after LPS challenge. Fatty acid composition of blood neutrophils and plasma was analyzed by HPLC.Results: Rats instilled with LPS presented a sixty three-fold increase in the number of neutrophils in BAL (from 0.5 × 10⁶ to 31.5 × 10⁶ cells), accompanied by increased levels of TNF- and IL-1 (p < 0.001), and a three-fold increase in ICAM-1 expression on vascular endothelium. The content of AA in blood neutrophils was reduced by 50%, whereas the level of PGE₂ in BAL was increased by 3.5 fold, without changes in the levels of LTB₄.Conclusions: These findings suggest that AA and PGE₂ are associated with LPS challenge.Received 19 October 2003; returned for revision 30 November 2003; accepted by N. Boughton-Smith 15 July 2004     

Sodium current in single myocardial mouse cells

Morphologically intact single myocardial cells of the adult mouse show a length of 132±20 m, a width of 21±5 , and a height of 10±4 m (all mean ± SD) and are brick-like in shape. A one suction pipette method is used for voltage clamp of those single cells. The determined time constant of capacitive current =35±14 s is very short. Series resistancer _s, membrane resistancer _m, and membrane capacityc _m are calculated to be 192±48 k, 6.1±1.1 M, and 186±92 pF (all mean ± SD), respectively. Assuming the specific unit membrane capacitance of 1 F/cm², a total membrane area of 1.86×10^–4 cm² is determined yielding a specific membrane resistanceR _m of 1,134 cm². Settling time of voltage clamp is 30 s. TTX-block of sodium current is described by 1:1 binding with aK _D value of 1.4×10^–6M. Using a reduced extracellular sodium concentration the maximum Na current is between 25 and 40 nA at voltages between –40 and –30 mV. Currents of between +20 and +30 mV reverse in an outward direction. Inward currents are approximated by a m³h model. The time constant of activation decreases from 0.7 ms at –60 mV to 0.12 ms at +20 mV. The time constant of inactivation falls from 9.1 ms at –60 mV to 0.6 ms at +20 mV.Steady state inactivationh is characterized by the half maximum valueV _H=–76.1±4.3 mV and the slope parameters=–6.3±1.1 mV (mean ± SD). A prepulse duration of 500 ms is essential for real steady state inactivation. Steady state activationm and inactivationh overlap each other defining a maximum window current at –65 mV.     

Circannual variation in the expression of β2-adrenoceptors on human peripheral mononuclear leukocytes (MNLs)

Summary Peripheral mononuclear leukocytes (MNLs) are widely used as a tissue model in studies of -adrenoceptor disturbances in hypertension and asthmatic diseases. The ₂-adrenoceptor density (B_max), however, depends not only on the gender of the person under study and on the time of day the blood specimens are obtained. Evidence is now reported for a circannual variation in the expression of ₂-adrenoceptor sites on peripheral MNLs. In male volunteers the 24-h mean was found to be highest in the men studied in April/May (1135±10 sites/cell) and decreased to 891±16 sites/cell in August and to 712±90 sites/cell in December (±SE,P<0.01 April/May compared to December). Concomitantly the circadian amplitude increased from 17.3%±6.4% of 24-h mean in April/May to 28.2%±1.4% of 24-h mean in August and to 34.2%±4.2% of 24-h mean in December (±SE,P<0.05, April/May compared to December). The circadian acrophase remained constant (190°±30° equivalent to 12 h 40 min±2 h 00 min, ±SE).Abbreviations MNLs Peripheral mononuclear leucocytes - B_{max 2}-Adrenoceptor density - ¹²⁵ICYP ¹²⁵Iodo-cyanopindolol - PEF Peak expiratory flow - SE Standard error - ANOVA Analysis of variance - M Circadian mesor - A Circadian amplitude - Circadian acrophase     

On the mechanism of β-adrenergic regulation of the Ca channel in the guinea-pig heart

Dose-response relations for the increase in the amplitude of Ca current (I _Ca) on external application of isoprenaline (ISP) and internally applied cyclic AMP (cAMP) or catalytic subunit of cAMP-dependent protein kinase (C subunit) were established in single ventricular cells of the guinea pig. An intracellular dialysis technique was used. The threshold concentration was for ISP 10^–9 M, for cAMP 3 M (pipette concentration to which 10^–5 M 3-isobutyl-1-methylxanthine was added) and for C subunit around 0.4 M (pipette concentration). The concentrations for the half-maximal effect were 3.7×10^–8 M (ISP), 5.0 M (cAMP) and 0.95 M (C subunit) and for the maximum effect 10^–6 M (ISP), 15–20 M (cAMP) and 3–4 M (C subunit). For all three agents the maximum increase in the Ca current density was similar (a factor of 3–4), suggesting that they converge on the same site of the Ca channel. Accordingly, the effects of cAMP and C subunit onI _Ca were non-additive to those of ISP. From these data the relationship both between concentrations of ISP and cAMP and between those of cAMP and active C subunit in terms of their effects onI _Ca could be estimated and were compared with those obtained in broken cell preparations.A competitive inhibitor of phosphorylation, 5-adenylyl-imidodiphosphate (5 mM), greatly reduced the effects of ISP and C subunit onI _Ca. Cell dialysis with 3 mM adenosine-5-(-thio)-triphosphate, which produces a dephosphorylationresistant phosphorylation, markedly potentiated the effects of ISP and cAMP onI _Ca.The results support the hypothesis that phosphorylation of a protein within, or close to, the Ca channel by cAMP-dependent protein kinase is the mechanism of -adrenergic stimulation.This work was supported by the Deutsche Forschungsgemeinschaft, SFB 38 (membranforschung), Projekt G, and H0579/6-2     

Characterization of the VIP- and secretin-stimulated adenylate cyclase system from human lung

The response of a crude particulate adenylate cyclase preparation from surgically removed human lung to guanine nucleotides, sodium fluoride, -adrenergic agonists, prostaglandins, vasoactive intestinal peptide (VIP), secretin, and [Val⁵]secretin was investigated. The enzyme activity increased 5, 10, and 9-fold, respectively, with GTP, Gpp(NH)p, and sodium fluoride. This activity was stimulated (in the presence as well as in the absence of added GTP) byd,l-isoproterenol,l-epinephrine andl-norepinephrine, the relative potency of these agonists being compatible with the existence of -adrenoceptors of the -adrenoceptors of the ₂ subtype. Prostaglandins E₁ and E₂, but not PGF₁ and PGF₂, stimulated the enzyme, PGE₁ being at least 10 times more potent than PGE₂. The biphasic pattern of stimulation of the same adenylate cyclase activity by VIP suggested the presence of high- and low-affinity VIP receptors coupled to the enzyme. This stimulation by VIP was not inhibited by secretin-(7–27). The stimulation of adenylate cyclase by secretin and [Val⁵]secretin was also biphasic, suggesting the coexistence of high- and low-affinity secretin receptors. Secretin-(7–27) was able to inhibit completely the secretin stimulation acting through high-affinity secretin receptors but exerted no effect on the stimulation operating through low-affinity secretin receptors, which might indicate that the latter receptors were in fact VIP-preferring receptors. [Val⁵]secretin was also used to differentiate these peptide receptors, since its properties were more VIP-like than those of secretin.Abbreviations VIP vasoactive intestinal peptide - Sn secretin - cyclic AMP cyclic adenosine 3:5-monophosphate - Gpp(NH)p guanosine 5-O-(2-3-imido)triphosphate - EGTA ethylene glycol bis (2-aminoethyl ether)-N,N,N,N-tetraacetic acid     

cAMP-dependent activation of small-conductance Cl− channels in HT29 colon carcinoma cells

The present study was performed to examine the conductance properties in the colon carcinoma cell line HT₂₉ and the activation of Cl^– channels by cAMP. A modified cell-attached nystatin patch-clamp technique was used, allowing for the simultaneous recording of the cell membrane potential (PD) and the conductance properties of the cell-attached membrane. In resting cells, PD was –56±0.4 mV (n=294). Changing the respective ion concentrations in the bath indicate that these cells possess a dominating K⁺ conductance and a smaller Cl^– conductance. A significant non-selective cation conductance, which could not be inhibited by amiloride, was only observed in cells examined early after plating. The K⁺ conductance was reversibly inhibited by 1–5 mmol/l Ba²⁺. Stimulation of the cells by the secretagogues isoproterenol and vasointestinal polypeptide (VIP) depolarized PD and induced a Cl^– conductance. Similar results were obtained with compounds increasing cytosolic cAMP: forskolin, 3-isobutyl-1-methylxanthine, cholera toxin and 8-bromoadenosine cyclic 3,5-monophosphate (8-Br-cAMP). VIP (1 nmol/l, n=10) and isoproterenol (1 umol/l, n=12) depolarized the cells dose-dependently and reversibly by 12±2 mV and 13±2 mV. The maximal depolarization was reached after some 20 s. The depolarization was due to increases in the fractional Cl^– conductance. Simultaneously the conductance of the cellattached membrane increased from 155±31 pS to 253±40 pS (VIP, n=4) and from 170±43 pS to 268±56 pS (isoproterenol, n=11), reflecting the gating of Cl^– channels in the cell-attached membrane. 5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mol/l) was without significant effects in resting and in forskolin-stimulated HT₂₉ cells. The agonist-induced conductance increase of the cell-attached nystatin patches was not paralleled by the appearance of detectable single-channel events in these membranes. These data suggest activation of small, non-resolvable Cl^– channels by cAMP.Supported by DFG Gr 480/10 and BMFT 01 GA 88/6     

Evidence for Na+/Ca2+ exchange in isolated smooth muscle cells: a fura-2 study

Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20–40 M or 4 M respectively, resulting in an estimated intracellular concentration of 100–200 M for quin-2 or 10–20 M fura-2 (free acid). On addition of 100 M carbachol or high K _o ⁺ (80 mM) depolarization, fura-2 loaded cells contracted (104±47 m,n=121 rest: 39±13 m,n=59 contracted) identically to control (103±35 m,n=232 rest: 39±16 m,n=89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca _i ²⁺ of fura-2 loaded cells was 100±18 nM (mean±SD,n=15) and was not significantly different from quin-2 loaded cells 107±26 nM (n=13). Treatment of fura-2 loaded cells with 100 M ouabain saline for 10–60 min progressively elevated the Ca _i ²⁺ to a mean of 266±83 nM (n=15). Reduction of Na _p ⁺ (96% Li⁺ replaced) significantly increased Ca _i ²⁺ to 317±77 nM (n=8). After pretreatment with ouabain (100 M), Na _o ⁺ replacement (Li⁺) increased Ca _i ²⁺ at a significantly faster rate [3.6 nM min^–1 (control) cf. 19.8 nM min^–1 (ouabain)].     

Chloride and sodium transport across bovine tracheal epithelium: Effects of secretagogues and indomethacin

The regulation of ion transport in bovine tracheal epithelium was studied in vitro. In the absence of exogenous midifiers of ion transport, average values for transepithelial electrical potential difference (_t), short-circuit-current (I _sc) and tissue resistance (R _t) were 35.4 mV (lumen negative), 5.4 Eq·h^–1·cm^–2 and 187 ·cm² respectively; net Cl secretion (3.2 Eq·h^–1·cm^–2) and net Na absorption (1.3 Eq·h^–1·cm²) accounted for 82% of theI _sc. Amiloride reduced (₁) andI _sc, and increasedR _t. The values of (_t),R _t andI _sc obtained following addition of theophylline, epinephrine or prostaglandin E₁ (PGE₁) were not different from control values. Theophylline aldo did not alter Na and Cl fluxes but it increased tissue cAMP content 3-fold. Indomethacin did not affect (_t) but it increasedR _t and net Na absorption, and decreasedI _sc and net Cl secretion; it did not significantly reduce tissue cAMP. When added to indomethacin-treated tissues, epinephrine restoredI _sc,R _t and Na and Cl fluxes to control levels and increased tissue cAMP 3-fold. Similary, when PGE₁ was added to indomethacin-treated tissues,I _sc andR _t were restored to control levels.We conclude that: (1) bovine tracheal epithelium, like its canine counterpart, absorbs Na and secretes Cl; the two tissues differ, however, in two ways: the spontaneous rate of Na absorption is higher in bovine trachea and the spontaneous rate of Cl secretion cannot be further increased in bovine trachea by secretagogues; (2) Cl secretion and Na absorption in bovine trachea are normally regulated by endogenous prostaglandins; (3) although cAMP may mediate changes in ion transport, a strict correlation between tissue cAMP content and Na and Cl transport rates is not evident; and (4) Na absorptive and Cl secretory rates are reciprocally related suggesting that both processes are present in the same cells.     

Localisation and characterisation of functional vasoactive intestinal peptide receptors in feline kidney

Specific ¹²⁵I-labelled vasoactive intestinal peptide (VIP) binding was determined in feline renal cortical and medullary plasma membranes. For the cortex, Scatchard analysis of the data resulted in a curvilinear plot with a high-affinity site K _0.5 of 8.4±2.6 nmol l^–1 (SE, n=6) and a second low-affinity site K _0.5 204 ± 16 nmol l^–1 with binding site concentrations (B _max) of 385±44.5 and 2710±181.3 fmol mg protein^–1 respectively. Conversely a similar analysis of the results obtained for outer medullary membranes gave a single site with a K _0.5 of 1.2±0.2 nmol l^–1 (SE, n=4) and B _max of 157.8±24.7 fmol mg^–1. Inner medullary membrane binding data. Gave a single site of lower affinity (K _0.5= 62.5±21.6 nmol l^–1; n=3). Structurally related peptides, glucagon and secretin, were ineffective (up to 1 mol l^–1) in displacing VIP from specific sites in both cortex and medulla. Porcine PHI 1–27 (a peptide having N-terminal histidine and C-terminal isoleucine) and a VIP antagonist [4-Cl-D-Phe⁶Leu¹⁷]VIP both displaced ¹²⁵I-VIP from cortical and medullary membrane binding sites with IC₅₀ values of 43.0 nmol l^–1 and 1.3 mol l^–1 (cortex) and 132.0 nmol l^–1 and 1.5 mol l^–1 (medulla) respectively. The localisation of specific VIP binding sites in feline kidney was investigated further by in vitro autoradiography. A high density of binding sites was visible at the cortico-medullary boundary as well as in the outer stripe of the outer medulla and in radial structures projecting into the cortex. There was a moderate density of binding sites in the superficial cortex. In addition the distribution of tubular VIP-sensitive adenylate cyclase was determined in microdissected nephron segments. In the presence of 10 mol l^–1 GTP, 1 mol l^–1 VIP effected marked stimulations over basal adenylate cyclase activity in medullary collecting tubules (4.7-fold), the bright and granular portions of the distal convoluted tubule (4.1- and 2.2-fold respectively) and in the pars recta of the proximal tubule (2.7-fold). Thus VIP-responsive adenylate cyclase has a discrete localisation in the feline nephron, which appears to correlate with specific binding sites as defined by autoradiography.     

Transmitter-induced changes of the membrane voltage of HT29 cells

The colonic carcinoma cell line HT₂₉ was used to examine the influence of agonists increasing cytosolic cAMP and Ca²⁺ activity on the conductances and the cell membrane voltage (V _m). HT₂₉ cells were grown on glass cover-slips. Cells were impaled by microelectrodes 4–10 days after seeding, when they had formed large plaques. In 181 impalements V _m was –51±1 mV. An increase in bath K⁺ concentration from 3.6 mmol/l to 18.6 mmol/l or 0.5 mmol/l Ba²⁺ depolarized the cells by 10±1 mV (n=49) or by 9±2 mV (n=3), respectively. A decrease of bath Cl^– concentration from 145 to 30 mmol/l depolarized the cells by 11±1 mV (n=24). Agents increasing intracellular cAMP such as isobutylmethylxanthine (0.1 mmol/l), forskolin (10 mol/l) or isoprenaline (10 mol/l) depolarized the cells by 6±1 (n=13), 15±3 (n=5) and 6±2 (n=3) mV, respectively. In hypoosmolar solutions (225 mosmol/l) cells depolarized by 9±1 mV (n=6). Purine and pyrimidine nucleotides depolarized the cells dose-dependently with the following potency sequence: UTP > ATP > ITP > GTP > TIP > CTP = 0. The depolarization by ATP was stronger than that by ADP and adenosine. The muscarinic agonist carbachol led to a sustained depolarization by 27±6 mV (n=5) at 0.1 mmol/l, and to a transient depolarization by 12±4 mV (n=5) at 10 mol/l. Neurotensin depolarized with a half-maximal effect at around 5 nmol/l. The depolarization induced by nucleotides and neurotensin was transient and followed by a hyperpolarization. We confirm that HT₂₉ cells possess Cl^–- and K⁺-conductive pathways. The Cl^– conductance is regulated by intracellular cAMP level, cytosolic Ca²⁺ activity, and cell swelling. The K⁺ conductance in HT₂₉ cells is regulated by intracellular Ca²⁺ activity.Supported by DFG Gre 480/10 and GIF Proj. no. I-86-100.10/ 88     

Phospholipase A2 (PLA2) activity in rabbit chondrocytes

The effects of recombinant interleukin-1 (rIL-1), recombinant tumor necrosis factor (rTNF) and two growth factors, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on PLA₂ activity and prostaglandin E₂ (PGE₂) release were investigated using rabbit chondrocytes. Cellular PLA₂ activity increased 2–10×above controls in the presence of 8×10^–12 M (5 U) rIL-1 or 5×10^–9 M rTNF after 20 hr incubation. PLA₂ activity remained constant with 1–50 ng/ml of either growth factor. PGE₂ release significantly increased (p<0.05) when the chondrocytes were incubated with rIL-1, bFGF and EGF alone, but not with rTNF above. These data suggest PLA₂ activity and PGE₂ release are not coordinately regulated in rabbit chondrocytes.     

Evidence for Na/H exchange and Cl/HCO3 exchange in A10 vascular smooth muscle cells

In the present study we used the pH sensitive absorbance of 5(and6)-carboxy-4,5-dimethylfluorescein to investigate intracellular pH (pH_i) regulation in A10 vascular smooth muscle cells: (1) The steady state pH_i in A10 cells averaged 7.01±0.1 (mean±SEM,n=26) at an extracellular pH of 7.4 (28 mM HCO₃/5% CO₂). (2) Removal of extracellular sodium led to an intracellular acidification of 0.36±0.07 pH-units (mean±SEM,n=8). (3) pH_i-Recovery after an acute intracellular acid load (by means of NH₄Cl-prepulse) was reversibly blocked by 1 mM amiloride and was dependent on the presence of sodium. The velocity of pH_i recovery increased with increasing sodium concentrations with an apparentK _m for external sodium of about 30 mM and aV _max of about 0.35 pH units/min. These findings are compatible with a Na/H exchanger being responsible for pH_i recovery after an acid load. (4) Removal of extracellular chioride induced an intracellular alkalinization of 0.23±0.03 pH-units (mean±SEM,n=10). The alkalinization was dependent on the presence of extracellular bicarbonate (5) Removal of chloride during pH_i recovery from an alkaline load (imposed by acetate prepulse) stopped and reversed pH_i backregulation. Chloride removal had no effect in the absence of bicarbonate or in the presence of 10^–4 M DIDS, suggesting that the effects were mediated by a Cl/HCO₃ exchanger. In conclusion we have demonstrated evidence for a Na/H exchanger and a Cl/HCO₃ exchanger in A10 vascular smooth muscle cells.Abbreviations used CDMF 5(and6)-carboxy-4,5-dimethylfluorescein - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - NMDG N-methyl-d-glucamine; pH_i, intracellular pH - pH_o extracellular pH - Mops 3-[N-Morpholino]propanesulfonic acid - Hepes 2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid - Tris Tris(hydroxymethyl)-aminomethane - EDTA ethylenediamine-tetraacetic acid - EGTA ethyleneglycol-bis-(-amino-ethylether)N,N-tetraacetic acid