An investigation of intrinsic buffering power in rat vascular smooth muscle cells

Intrinsic buffering power ( _i) has been measured in vascular strips and single cells from rat mesenteric artery. Intracellular pH (pH_i) regulation was inhibited to prevent overestimation of _i due to acid extrusion or entry via regulatory processes. At resting values of pH_i (7.0–7.2), a mean value of 41±4 mM/pH unit for _i was found. _i increased approximately fivefold from 30 to 150 mM/pH unit over the pH_i range 7.5–6.5. The mean data relating _i to pH_i could be described by relating _i to buffer concentrations and pK _a. This gave a value of 310 mM for buffer concentration and a pK _a of 6.0. As changes in pH_i are known to have marked effects on vascular tone then the increase in _i as pH_i falls may be considered as a means of attenuating pH_i decreases, before pH regulation restores pH_i to resting levels.     

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.     

A new double-barrelled,ionophore-based microelectrode for chloride ions

A new Cl^– selective microelectrode based on the ionophore 5,10,15,20-tetraphenyl-21H,23H-porphin manganese(III) chloride is presented which discriminates better against HCO ₃ ^– and several organic anions than electrodes containing the Corning 477913 ion-eschanger. Using a redesigned construction procedure, fine-tip double-barrelled microelectrodes were produced which had slopes of –52.4±0.6 mV (SE,n=24), resistances of about 7·10¹¹ and a selectivity coefficient logK _{Cl, HCO3} ^pot of –1.40±0.03. Some electrodes showed a small unexplained sensitivity to pH>7.6. When used to puncture cells of isolated S3 segments of rabbit renal proximal tubule during perfusion with HCO ₃ ^– Ringer solution, the electrodes gave a membrane potential of –69.8±1.5 mV and an intracellular Cl^– activity, [Cl^–]_i, of 35.3±2.6 mmol/l. Upon switching bath and lumen perfusions to Cl^–-free solutions the residual [Cl^–]_i dropped to 1.20±0.03 mmol/l, while in similar measurements with ion-exchanger electrodes the residual [Cl^–]_i dropped only to 10.9±0.5 mmol/l. These observations demonstrate the superiority of the new electrode and prove that previously determined high [Cl^–]_i values in Cl^–-free ambient solutions reflect interference problems rather than non-exchangeable intracellular chloride.     

Small-conductance chloride channels activated by calcium on cultured endocrine cells from mammalian pars intermedia

Porcine intermediate lobe (IL) endocrine cells maintained in primary culture have been studied using patch-clamp derived configurations to record unitary activity on outside-out vesicles. Solutions were devised so as to record Cl current in isolation and to fix cytoplasmic Ca concentration [Ca]_i between 0.1 M and 3 M. Between [Ca]_i 0.5 and 1 M, the chloride permeability was restricted to single events with a small amplitude, that varied linearly with the membrane potential. Mean slope conductance of this chloride channel was 2.5 pS. Single channel analysis yielded two mean open time values of 10 and 55 ms at –80 mV. Relaxations of chloride currents on outside-out patches was examined at different [Ca]_i. Relaxation was negligible at 0.15 M [Ca]_i, whereas at higher [Ca]_i, the current exhibited relaxation in response to voltage jumps the kinetic of which could be fitted by two exponentials. At 0.5 M [Ca]_i, the fast relaxation time constant was shown to be voltage insensitive with a value of about 10 ms. The slow relaxation time constant had a mean value of 75 ms at –60 mV and increased with membrane depolarization with a twofold change over 120 mV. Another voltage effect was to favour the slow opening mode at the more depolarized potentials: the ratio of fast to slow relaxations being 5:1 at –60 mV as compared to 11 at +80 mV). Finally the estimated probability of opening (p _o) linearly increased with voltage.p _o displayed a bell-shaped dependence on [Ca]_i, so that full activation of the channels was not achieved.     

Cardiac Purkinje fibres

Summary The influence of intracellular calcium concentration [Ca²⁺] _i on the steady state membrane currentsi was studied in a range of clamp potentials between –20 and –100 mV. Injection of CaCl₂ or Ca-EGTA (pCa 6) increasedi whereas injection of K-EGTA diminished it. The changes i were attributed to a change in steady state potassium conductance, g_K, by four arguments: i was restricted to potentials negative to –20 mV and depended on clamp potential in an inward rectifying manner. i displayed a reversal potential, E_rev, which followed log [K⁺]₀ with 60 mV for a tenfold change. Since E_rev obtained during Ca injection agreed with E_rev observed during EGTA injection the potassium driving force had to be constant. Wheng _K was blocked by superfusion with 20 mM Cesium neither CaCl₂ nor K-EGTA injection modifiedi .Supported by SFB 38, Membranforschung, project G2     

An electrophysiological study of angiotensin II regulation of Na-HCO3 cotransport and K conductance in renal proximal tubules

The effect of picomolar concentrations of angiotensin II (AII) was investigated in isolated perfused rabbit renal proximal tubules using conventional or pH-sensitive intracellular microelectrodes. Under control conditions cell membrane potential (V _b) and cell pH (pH_i) averaged –53.8±1.9 mV (mean±SEM,n=49) and 7.24±0.01 (n=10), respectively. AII (at 10^–11 mol/l), when applied from the bath (but not when applied from the lumen perfusate), produced the following effects: approximately 85% of the viable tubules responded with a small depolarization (+ 5.5±0.4 mV,n=43) which was accompanied in half of the pH_i measurements by a slow acidification (pH_i=–0.03±0.01,n=5). The remaining 15% responded with a small hyperpolarization (V_b=–3.1±0.4 mV,n=6). All changes were fully reversible and repeatable. Experiments with fast changes in bath HCO₃ or K concentrations, as well as measurements of the basolateral voltage divider fraction in response to transepithelial current flow, explain these observations as stimulation of a basolateral Na-HCO₃ cotransporter and of a basolateral K conductance. Both counteract in their effect onV _b, but can be individuated by blocker experiments with 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) and barium. Both the stimulation of Na-HCO₃ cotransport and the stimulation of the K conductance may result from down-regulation of the level of cyclic adenosine monophosphate in the cell.     

Tubular transport processes in proximal tubules of hypothyroid rats. Lack of relationship between thyroidal dependent rise of isotonic fluid reabsorption and Na+−K+-ATPase activity

Hypothyroid rats reconstituted with 10 g/kg b.w. per day of tri-iodothironine (T₃) for 4 days resulted in normal free T₃ and TSH levels. FT₃ levels were: 0.53±0.3 pg/ml in hypothyroid rats; 2.78±1.21 pg/ml in hormone reconstituted rats and 2.90±0.90 pg/ml in euthyroid rats. TSH levels were 3,508±513 g/ml in hypothyroid rats; 1,008±204 g/ml in reconstituted rats and 270±184 ng/ml in euthyroid rats.When hypothyroid rats were reconstituted with 50 g T₃/kg b.w. per day, TSH levels were nearly normal after 4 days (1,157±621 ng/ml). However FT₃ levels after 1–4 days were always higher than in euthyroid rats.Hypothyroid rats show a decrease in isotonic fluid reabsorption (J _v) in the proximal tubule (1.50±0.08 versus 4.96±0.23 10^–2 nl·mm^–1·s^–1 in euthyroid animals). 1 day after T₃ (10 g/kg b.w./day) injectionJ _v was increased significantly to 2.05±0.20 10^–2 nl·mm^–1·s^–1 and continued to increase during 4 days of T₃ reconstitution.When 50 g T₃/kg b.w./day was used,J _v increased to 2.75±0.07 after 1 day and to 3.10±0.42 10^–2 nl·mm^–1·s^–1 after 4 days.J _v was never reaching a value close to that of euthyroid rats because the tubular radius in hypothyroid rats (14.7±1.8 m) is less than that of euthyroid rats (19.2±0.5 m). The radius in hypothyroid rats treated with T₃ was unchanged over a 4 day course with either high or low doses of T₃.Na⁺–K⁺-ATPase activity was found to be 2.91±0.16 M P_i/h×mg protein in homogenates of kidney cortex from hypothyroid rats. Treatment of hypothyroid rats with 10 g or 50 g of T₃ resulted in an initial decrease in ATPase activity, followed by an increase to base level in hypothyroid rats with 10 g and a significantly higher level with 50 g. This decrease in ATPase activity was contrasted to the increase inJ _v.These data indicate that there is a dissociation between the effects of physiological doses of thyroid hormones on proximal tubular reabsorption and the effects of T₃ on Na⁺–K⁺-ATPase activity of kidney cortex. This leads to question the relationship between sodium transport and ATPase activity under physiological doses of thyroid hormones. An early effect of physiological doses of thyroid hormones on brush border Na⁺ permeability is suggested.     

Action potential and membrane currents of single pacemaker cells of the rabbit heart

Single, viable pacemaker cells were isolated from sinoatrial (S-A) and atrioventricular (A-V) nodes by treating with collagenase. In normal Tyrode solution containing 1.8 mM Ca²⁺, these pacemaker cells had a round configuration and contracted rhythmically at a frequency of about 150–260/min. The amplitude, duration, and maximum rate of rise of the spontaneous action potentials recorded using patch clamp electrodes were similar to those obtained from multicellular preparations. Amplitudes of the recorded membrane current were normalized with reference to the surface area of the cell by assuming the cell shape as a plane oblate spheroid. The membrane resistance of the isolated nodal cells was 14.9±4.0 k·cm² (n=12) at about –35 mV and the membrane capacitance was 1.30±0/24 F/cm² (n=18). The inactivation time course of the slow inward current,i _si, was fitted with a sum of two exponentials with time constants of 6.7±0.6 ms and 46.6±15.3 ms (n=4) at +10 mV. The amplitude ofi _si peaked at 0+10 mV in the current-voltage relationship and was 18.2±8.4 A/cm². The potassium current,i _K was activated in the voltage range positive to –50 mV and was saturated at about +20 mV. The amplitude of the fully-activatedi _K at –40 mV was 3.3±1.4 A/cm² (n=10) and showed an inward-going rectification. The activation of the hyperpolarization-activated current was observed at potentials negative to –70 mV in seven of 14 experiments. The current density and membrane capacitance calculated could be overestimated and the membrane resistance underestimated, because of the presence of caveolae on the cell surface. However, these data give the nearest possible estimates of the electrical constants in the nodal cells, which cannot be measured accurately in the conventional multicellular preparations.     

Zinc uptake by proximal cells isolated from rabbit kidney: Effects of cysteine and histidine

The aim of this study was to characterize the mechanisms of zinc transport in proximal cells isolated from rabbit kidney cortex. Uptakes of ⁶⁵Zn were assessed under initial rate conditions, after 0.5 min of incubation. The kinetic parameters obtained at 20°C were a K _m of 15.0±1.5 M, a J _max of 208.0±8.4 pmol min^–1 (mg protein)^–1, and an unsaturable constant of 0.259±0.104 (n=8). Cadmium competitively inhibited the zinc uptake, with a K _i value of 13.0±2.8 M, while zinc competitively inhibited ¹⁰⁹Cd uptake by isolated cells. Cysteine and histidine stimulated zinc transport at an amino acidzinc molar ratio ranging from 11 to 81. This stimulation was not observed in the absence of a sodium gradient. At a molar ratio greater than 161 (i.e., 400 M cysteine or histidine and 25 M Zn), there was evidence of inhibition. These data suggest that zinc enters renal proximal cells (a) as a free ion via a saturable carrier-mediated process or an unsaturable pathway and (b) complexed with cysteine or histidine, by means of a sodium/amino acid cotransport mechanism.     

The Ba2+ block of the ATP-sensitive K+ current of mouse pancreaticβ-cells

We studied the block of whole-cell ATP-sensitive K⁺ (K_ATP) currents in mouse pancreatic-cells produced by external Ba²⁺. Ba²⁺ produced a time- and voltage-dependent block of K_ATP currents, both the rate and extent of the block increasing with hyperpolarization. With 5.6 mM [K⁺]_o, the relationship between the steady-state KATP current and [Ba²⁺]_o, was fit by the Hill equation with aK _d of 12.5 ± 2.8 M at –123 mV and of 0.18 ± 0.02 mM at –62 mV The Hill coefficient (n) was close to 1 at all potentials indicating that binding of a single Ba²⁺ ion is sufficient to block the channel. When [K⁺]_o was raised to 28 mM the K_d was little changed (12.4 ± 4.1 gM at –123 mV 0.27 ± 0.05 mM at –62 mV) and n was unaffected, suggesting that K⁺ does not interact with the Ba²⁺ binding site. The kinetics of Ba²⁺ block were slow, 10 M Ba²⁺ blocking the K_ATP current with a time constant of 20 ms at –123 mV in 28 mM [K⁺]_o. The blocking rate constant was calculated as 1.7 mM^–1 ms^–1 and the unblocking rate as 0.02 ms^–1, at –123 mV The data are discussed in terms of a model in which Ba²⁺ binds to a site at the external mouth of the channel to inhibit the K_ATP channel.     

Some properties of the UL-FS 49 block of the hyperpolarization-activated current (i f) in sino-atrial node myocytes

Block of the hyperpolarization-activated pacemaker current (i _f) by the bradycardic agent UL-FS 49 was studied in isolated sino-atrial (SA) node myocytes. Using repetitive activation/deactivation protocols, micromolar concentrations of UL-FS 49 blockedi _f in a dose-dependent fashion. Block development was slow, with time constants decreasing with drug concentration and ranging from 25.8 s at 10 M to 75.5 s at 1 M UL-FS 49. Block did not develop in cells held at –35 mV, at which voltagei _f channels are closed, indicating that channels must open before blocking occurs. Apparently in contrast with the requirement of negative voltages for block development, block was relieved by hyperpolarization with a time course slower than current kinetics. Due to the hyperpolarization-induced block relief, current/voltage (I/V) relations in the presence of UL-FS 49 displayed inward-going rectification. Experimental data fitted the hypothesis that UL-FS 49 behaves as an open channel blocker of single-ioni _f channels. Block occurs within the pore, at a distance of about 39% of the membrane thickness from its internal side.     

Effects of serotonin on electrical properties of Madin-Darby canine kidney cells

The present study has been performed to test for the influence of serotonin on the potential difference across the cell membrane (PD) of Madin-Darby canine kidney (MDCK)-cells. Under control conditions PD averages –48.6±0.6 mV (n=98). Increasing extracellular potassium concentration from 5.4 to 10 and 20 mmol/l depolarizes the cell membrane by +6.3±0.6 mV (n=6) and +14.1±1.0 mV (n=12), respectively. The cell membrane is transiently hyperpolarized to –67.8±0.8 mV (n=63) by 1 mol/l serotonin. In the presence of serotonin, increasing extracellular potassium concentration from 5.4 to 20 mmol/l depolarizes the cell membrane by +26.4±1.0 mV (n=11). 1 mmol/l barium depolarizes the cell membrane by +15.7±1.3 mV (n=17) and abolishes the effect of step increases of extracellular potassium concentration from 5.4 to 10 mmol/l. In the presence of barium, serotonin leads to a transient hyperpolarization by –26.3±1.0 mV (n=16). During this transient hyperpolarization, the cell membrane is sensitive to extracellular potassium concentration despite the continued presence of barium. 10 mol/l methysergide hyperpolarize the cell membrane by –7.2±2.0 mV (n=6). In the presence of 10mol/l methysergide, the effect of serotonin is virtually abolished (+0.4±0.9 mV,n=6). 1 mol/l ketanserin, a 5-HT₂ receptor blocking agent, ICS 205-930, a 5-HT₃ receptor blocking agent, and phentolamine, an unspecific -receptor blocking agent, do not significantly modify the effect of serotonin. In the nominal absence of extracellular calcium, the effect of serotonin is markedly reduced. In conclusion, serotonin hyperpolarizes MDCK-cells by increasing apparent potassium conductance. This effect is transmitted by 5-HT₁ receptors and depends on extracellular calcium.     

Electrical properties of gap junction channels in guinea-pig ventricular cell pairs revealed by exposure to heptanol

Cell pairs were isolated from adult guinea pig ventricles to study the electrical properties of gap junction channels. The experiments involved a double voltage-clamp approach and whole-cell, tight-seal recording. Heptanol decreased the intracellular current, I _n, in a dose-dependent fashion. Before complete uncoupling, I _n showed fluctuations suggesting the operation of gated channels. In the presence of 3 mM heptanol, I _n showed quantal steps arising from spontaneous opening and closing of single channels. The IV-relationship of the channels was linear (range: ±95 mV). Analysis of current records revealed the following singlechannel conductances, _n: Mean value = 37 pS; median value = 33 pS. _n was insensitive to the non-junctional membrane potential (range: –90 to +10 mV). 3 mM ATP^4– in the pipette solution had no effects on _n, 6 mM ATP^4– produced a small decrease, and 6 mM ATP+0.1 mM cAMP^– an increase in _n. Channel transitions from closed to open state were variable (range of apparent time constants: 2.5–32 ms; mean: 11 ms).     

Volume flow,hydraulic conductivity and electrical properties across bovine tracheal epithelium in vitro: Effect of histamine

Volume flow (J _v), potential difference (), shortcircuit current (i ₀) and electrical resistance (R) were measured simultaneously across bovine tracheal epithelium in vitro. Under basal conditions, with no applied hydrostatic or osmotic pressure gradient (P=0, =0), no spontaneousJ _v was observed. was 31±2 mV (lumen negative),i ₀ 161±8 A cm^–2 andR 202±9 cm²,n=50. When a was applied, by adding 20–80 mM sucrose into the medium bathing either the luminal or the serosal side of the tissue, a linear relationship was found between andJ _v toward the lumen or toward the serosa. The apparent hydraulic conductivity (apparentL _p) was 4.6–4.910^–6 cm s^–1 atm^–1. Histamine 10^–4 M did not induce any spontaneousJ _v under basal conditions and had no effect oni ₀ nor onR. However, histamine caused a 100% increase inJ _v elicited by sucrose gradients. It was concluded that histamine exerts a selective action on the hydraulic conductivity of bovine tracheal epithelium. Experiments using H₁-receptors antagonists (diphenhydramine, dimetindene, chloropyramine) and H₂-antagonists (cimetidine, metiamide) or a H₂-agonist (impromidine) showed that the increase ofL _p induced by histamine was mediated via H₂-receptors.Supported by the Swiss National Foundation (SNF), grant no. 3.5880.79     

Sodium current kinetics in freshly isolated neostriatal neurones of the adult guinea pig

Neurones of the neostriatum were freshly dispersed from the adult guinea pig brain. A fast, transient inward Na⁺ current (I _Na) was analysed using the whole-cell patch-clamp technique. Upon depolarizations, I _Na developed with a sigmoidal time course, which was described by m ³ kinetics. I _Na showed an activation threshold of about –60 mV, a peak current at –30 to –20 mV, and a reversal of polarity at +60 mV. The steady-state activation (m) curve for I _Na had a slope factor of about 9 mV with a mid-point potential of about –26 mV. The voltage dependence of the activation time constant, _m , had a bell-shaped configuration with a maximum value at –60 mV. The forward rate constant for I _Na activation ( _m ) increased as the membrane was depolarized (about 9 mV for a change in the rate constant by a factor of e) in the range between –50 mV and –20 mV. Conversely, the backward rate constant ( _m) decreased as the membrane was depolarized (about 31 mV for an e-fold change). The steady-state inactivation (h ) curve was well expressed by the Boltzmann's equation with a half-inactivation potential of –62 mV and a slope parameter of 6 mV. The time course of I _Na decay followed a second-order process, whereas the recovery from inactivation was described as a first-order process. The _h curve showed a bell-shaped configuration with a maximum value at –60 mV. The forward rate constants ( _h ) decreased as the membrane was depolarized (about 17 mV for an e-fold change) in the range between –50 mV and –20 mV. The backward rate constants ( _h) increased as the membrane was depolarized (about 10 mV for an e-fold change). There was a significant overlap between m and h curves, suggesting a steady influx of Na⁺ (window current).     

β-Adrenoceptor stimulation activates large-conductance Ca2+-activated K+ channels in smooth muscle cells from basilar artery of guinea pig

We studied the effect of isoproterenol on the Ca²⁺-activated K⁺(BK) channel in smooth muscle cells isolated from the basilar artery of the guinea pig. Cells were studied in a whole-cell configuration to allow the clamping of intracellular Ca²⁺ concentration, [Ca²⁺]_i. Macroscopic BK channel currents were recorded during depolarizing test pulses from a holding potential (V _H) of 0 mV, which was used to inactivate the outward rectifier. The outward macroscopic current available from aV _H of 0 mV was highly sensitive to block by external tetraethylammonium·Cl (TEA) and charybdotoxin, and was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. With [Ca²⁺]_i between 0.1 and 1.0 M, 0.4 M isoproterenol increased this current by 58.6±17.1%, whereas with [Ca²⁺]_i at 0.01 M a sixfold smaller increase was observed. With [Ca²⁺]_i0.1 M, 100 M dibutyryl-adenosine 3:5: cyclic monophosphate (cAMP) and 1 M forskolin increased this current by 58.5±24.1% and 59.7±10.3%, respectively. The increase with isoproterenol was blocked by 4.0 M propranolol extracellularly, and by 10 U/ml protein kinase inhibitor intracellularly. Single-channel openings during depolarizing test pulses from aV _H of 0 mV recorded in the whole-cell configuration under the same conditions (outside-outwhole-cell recording) indicated a slope conductance of 260 pS. In conventional outside-out patches, this 260-pS channel was highly sensitive to block by external TEA, and in inside-out patches, its probability of opening was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. Outside-out-whole-cell recordings with [Ca²⁺]_i0.1 M indicated that 100 M dibutyryl-cAMP increased the probability of opening of the 260-pS channel by 152±115%. In inside-out patches, the catalytic subunit of protein kinase A increased the probability of opening, and this effect also depended on [Ca²⁺]_i, with a 35-fold larger effect observed with 0.1–0.5 M Ca²⁺ compared to 0.01 M Ca²⁺. We conclude that the BK channel in cerebrovascular smooth muscle cells can be activated by-adrenoceptor stimulation, that the effect depends strongly on [Ca²⁺]_i, and that the effect is mediated by cAMP-dependent protein kinase A with no important contribution from a direct G-protein or phosphorylation-independent mechanism. Our data indicate that the BK channel may participate in-adrenoceptor-mediated relaxation of cerebral vessels, although the importance of this pathway in obtaining vasorelaxation remains to be determined.     

Demonstration of A-currents in pancreatic islet cells

Voltage-activated K⁺ currents resistant to TEA but blockable by 4-AP were recorded from mouse pancreatic islet cells. These currents first become observable during depolarizations to voltages more positive than –40 mV, reaching a peak amplitude of 120±34 pA at +6 mV (n=4), display rapid turn on (=3.3±1.1 ms at +6 mV) and inactivate completely within 250 ms (=65±5 at +6 mV). The current is subject to steady-state inactivation. The midpoint (V _h) of the inactivation curve (h) was observed at –72±2 mV. The properties of this current resemble those reported for the A-current in neurons.     

Contribution of prostaglandins in hypoxia-induced vasodilatation in isolated rabbit hearts. Relation to adenosine and KATP channels

The mechanism of hypoxia-induced coronary vasodilatation was studied in isolated, saline-perfused rabbit hearts under constant flow conditions. Reduction in the perfusion solution PO₂ (from 520±6 to 103±9 mm Hg) under control conditions halved the coronary resistance and was accompanied by a significant release of the prostaglandin (PG) 6-keto-PGF₁ (from 1.8±0.3 to a maximum of 4.4±0.9 pmol min^–1 g^–1). The cyclooxygenase inhibitor, diclofenac (1 M), blocked the release of PGI₂ and reduced hypoxia-induced vasodilatation (from 47±8% to 25±5%, P<0.05). The relative contribution of adenosine, prostaglandins, and adenosine triphosphate (ATP)-sensitive K⁺ channel (K_ATP channel) activation in hypoxia-induced vasodilatation was assessed by comparing the differential change (control response minus response after treatment) in coronary perfusion pressure (CPP) during infusion of 8-phenyltheophylline (8-PT), diclofenac, and glibenclamide, respectively. The differential change in CPP with 8-PT and diclofenac given together (–48 ±7%) was found to be equivalent to the sum of their respective effects (–24±7 and –19±4%, respectively). Glibenclamide (0.3 M) reduced significantly hypoxia-induced vasodilatation (differential change in CPP of –27±6%) as well as the dilator response to 10 M adenosine and to the stable PGI₂-analogue, iloprost. Forskolin-induced coronary vasodilatation in arrested hearts was slightly, but significantly, reduced by glibenclamide. Our results suggest that both cyclooxygenase products and adenosine, acting independently and concomitantly, contribute to the dilator response of coronary resistance vessels to hypoxia, in part through the activation of K_ATP channels. K_ATP channel activation by prostacyclin and adenosine may involve both cyclic adenosine monophosphate-dependent and independent pathways.     

Untersuchungen zur Stabilit?t, Empfindlichkeit und Linearit?t der pCO2-Elektrode

Zusammenfassung Es wird eine spaltlose pCO₂-Elektrode beschrieben, die auch im Routinebetrieb eine gute Stabilität und Reproduzierbarkeit bei schneller Einstellzeit zeigt.Der mittlere Fehler der Einzelmessung beträgt ±0,2 bis ±1,2 mm Hg pCO₂ im Bereich von 30–90 mm Hg pCO₂. Die Einstellzeit ist zwischen 60 und 90 sec (100%) unter Verwendung von 0,001 M NaHCO₃-Lösung, 30 Nylonfäden und 12 Teflonmembran. Die Eichkurve für Blut und Gas ist gleich. Sie ist im Bereich von 10–90 mm Hg pCO₂ (pH=6,2–7,1) linear. Die Steilheit beträgt S=0,92–0,95. Die Drift ist 0,1 mV/Std.Diese Eigenschaften wurden im wesentlichen durch zwei Verbesserungen erreicht: 1. Zwischen pH-empfindlicher Zone und Gehäusekante wurde eine pH-unempfindliche Zone angeordnet, in der die Äquilibierung der NaHCO₃-Lösung genauso schnell erfolgt, wie unter der pH-empfindlichen Membran. Dadurch wird der störende Einfluß der Elektrolytbrücke ausgeschaltet (spaltlose Elektrodenform). Diese Anordnung bewährt sich auch bei anderen Meßelektroden, die nach einem ähnlichen Prinzip arbeiten. 2. Es wurden nur Materialien verwandt, die den pK der NaHCO₃-Lösung nicht beeinflussen.Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.Kardiologische Abteilung der Universität Utrecht (Direktor: Prof. R. L. J. van Ruyven).     

Go transduces GABAB-receptor modulation of N-type calcium channels in cultured dorsal root ganglion neurons

High-voltage-activated (HVA) calcium channel currents (I _Ba) were recorded from acutely replated cultured dorsal root ganglion (DRG) neurons. I _Ba was irreversibly inhibited by 56.9±2.7% by 1 M -conotoxin-GVIA (-CTx-GVIA), whereas the 1,4-dihydropyridine antagonist nicardipine was ineffective. The selective -aminobutyric acid_B (GABA_B) agonist, (–)-baclofen (50 M), inhibited the HVA I _Ba by 30.7±5.4%. Prior application of -CTx-GVIA completely occluded inhibition of the HVA I _Ba by (–)-baclofen, indicating that in this preparation (–)-baclofen inhibits N-type current. To investigate which G protein subtype was involved, cells were replated in the presence of anti-G protein antisera. Under these conditions the antibodies were shown to enter the cells through transient pores created during the replating procedure. Replating DRGs in the presence of anti-G_o antiserum, raised against the C-terminal decapeptide of the G _o subunit, reduced (–)-baclofen inhibition of the HVA I _Ba, whereas replating DRGs in the presence of the anti-G_i antiseram did not. Using anti-G _o antisera (12000) and confocal laser microscopy, G _o localisation was investigated in both unreplated and replated neurons. G _o immunoreactivity was observed at the plasma membrane, neurites, attachment plaques and perinuclear region, and was particularly pronounced at points of cell-to-cell contact. The plasma membrane G _o immunoreactivity was completely blocked by preincubation with the immunising G_o undecapeptide (1 g · ml^–1) for 1 h at 37° C. A similar treatment also blocked recognition of G _o in brain membranes on immunoblots. These results provide evidence that GABA_B inhibition of N-type calcium channels in acutely replated DRGs occurs via G _o.