W.A. Large

Potassium, chloride and non-selective cation conductances opened by noradrenaline in rabbit ear artery cells.

1. The action of noradrenaline on cells isolated from the rabbit ear artery was studied with the whole‐cell configuration of the patch clamp technique. In normal potassium‐containing solutions at a holding potential of ‐50 mV noradrenaline elicited either outward, inward or mixed outward and inward currents. These responses were blocked by the alpha‐adrenoceptor antagonist, phentolamine (10(‐6) M). 2. The outward current occurred as a consequence of an increase in membrane conductance and the reversal potential was close to the potassium equilibrium potential (EK). It was possible to record outward currents without external calcium but not when the concentration of EGTA in the pipette was increased to 10 mM or when potassium was absent from the pipette solution. It is concluded that the outward current evoked by alpha‐adrenoceptor stimulation is produced by a calcium mediated increase in potassium conductance. 3. The ionic basis of the inward current was investigated in potassium‐free external and pipette solutions. When sodium chloride was the major constituent of the external and pipette solutions the reversal potential (Er) of the noradrenaline‐induced current was 0.63 mV, close to ENa and ECl. 4. When most of the external sodium chloride was replaced by sucrose Er was intermediate between ENa and ECl but had shifted significantly towards ENa. Further ion substitution experiments suggest that noradrenaline increased the membrane conductance to both anions and cations. 5. When the current was carried predominantly by anions, depolarizing steps (from ‐50 mV) produced outward current relaxations with a time constant of about 40 ms. Bath‐applied caffeine also produced a membrane current which rectified in the outward direction. 6. When the response to noradrenaline was mediated mainly by cations, the relationship between the membrane current and clamp potential was non‐linear and the amplitude of the currents at potentials positive to ‐50 mV became disproportionately smaller. In addition on repolarization to ‐50 mV the instantaneous current was followed by an inward relaxation. 7. In high external barium solution noradrenaline evoked a membrane current with a reversal potential much more positive than ENa or ECl. This current was recorded in the presence of 10(‐5) M‐nifedipine and diltiazem and in 10(‐4) M‐cadmium which suggests that the voltage‐dependent calcium channel is not implicated in the generation of the non‐selective cation current to noradrenaline.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of niflumic acid on noradrenaline‐induced contractions of the rat aorta

1 The effects of niflumic acid, an inhibitor of calcium‐activated chloride channels, were compared with the actions of the calcium channel antagonist nifedipine on noradrenaline‐evoked contractions in isolated preparations of the rat aorta. 2 The cumulative concentration‐effect curve to noradrenaline (NA) was depressed by both nifedipine and niflumic acid in a reversible and concentration‐dependent manner. The degree of inhibition of the maximal contractile response to NA (1 μm) produced by 10 μm niflumic acid (38%) was similar to the effect of 1 μm nifedipine (39%). 3 Contractions to brief applications (30 s) of 1 μm NA were inhibited by 55% and 62% respectively by 10 μm niflumic acid and 1 μm nifedipine. 4 In the presence of 0.1 μm nifedipine, niflumic acid (10 μm) produced no further inhibition of the NA‐evoked contractions. Thus, the actions of niflumic acid and nifedipine were not additive. 5 In Ca‐free conditions the transient contraction induced by 1 μm NA was not inhibited by niflumic acid (10 μm) and therefore this agent does not reduce the amount of calcium released from the intracellular store or reduce the sensitivity of the contractile apparatus to calcium. 6 Niflumic acid 10 μm did not inhibit the contractions produced by KCl (up to 120 mM) which were totally blocked by nifedipine. Contractions induced by 25 mM KCl were completely inhibited by 1 μm levcromakalim but were unaffected by niflumic acid. 7 It was concluded that niflumic acid produces selective inhibition of a component of NA‐evoked contraction which is probably mediated by voltage‐gated calcium channels. These data are consistent with a model in which NA stimulates a calcium‐activated chloride conductance which leads to the opening of voltage‐gated calcium channels to produce contraction.

NORADRENALINE-EVOKED CATION CONDUCTANCE RECORDED WITH THE NYSTATIN WHOLE-CELL METHOD IN RABBIT PORTAL VEIN CELLS

1. Noradrenaline‐evoked currents were studied with the perforated patch technique (nystatin in the patch pipette) in freshly dispersed rabbit portal vein cells. 2. With potassium‐containing solutions noradrenaline produced an inward current at ‐50 mV and an outward current (IK(Ca] at 0 mV. In potassium‐free conditions noradrenaline elicited two distinct membrane currents which could be differentiated according to their sensitivity to the presence of caffeine in the bathing solution. One current was blocked whereas the second response was unaffected by caffeine. 3. The reversal potential (Er = ‐2.0 mV) of the caffeine‐sensitive current was altered when external chloride was replaced with more permeant anions but Er was not changed when external sodium was replaced by Tris. Therefore the caffeine‐sensitive current appears to be a calcium‐activated chloride current (ICl(Ca]. 4. Er (+6.0 mV) of the caffeine‐insensitive current was not altered by anion substitution but was changed when external sodium was replaced by Tris and barium ions. Thus the caffeine‐insensitive conductance is a non‐selective cation current (Icat). 5. When external NaCl was replaced by BaCl2, Er was shifted to more positive potentials which suggests that the cation conductance is more permeable to barium than to sodium. Icat was not affected by 10(‐6) M‐nifedipine. 6. The steady‐state current‐voltage relationship was linear between ‐50 and +50 mV for ICl(Ca) but the cation conductance mechanism displayed pronounced inward rectification and little outward current flowed across the membrane at positive potentials. 7. Caffeine (which releases calcium from internal stores) and the calcium ionophore ionomycin, which are expected to increase intracellular calcium concentration, evoked ICl(Ca) but not Icat. Thus ICl(Ca) but not Icat can be activated directly by an increase in intracellular calcium concentration. 8. When calcium was removed from the bathing solution the amplitude of Icat was not altered at early times in Ca(2+)‐free conditions but was abolished after 10 min. Icat was readily activated by noradrenaline in the presence of ionomycin, which inactivated ICl(Ca). A permissive role for calcium in the generation of Icat is suggested. 9. The times between the application of noradrenaline and the onset of the chloride and cationic conductances were similar (0.75 and 0.9 s respectively). In contrast the rise time (3.9 s) and half‐decay time (11 s) of Icat were much longer than the corresponding values of ICl(Ca) (respectively 1.9 and 2 s). In high‐barium solution noradrenaline sometimes evoked a response that lasted for up to 10 min.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibitory action of niflumic acid on noradrenaline- and 5-hydroxytryptamine-induced pressor responses in the isolated mesenteric vascular bed of the rat

The effects of niflumic acid, an inhibitor of calcium‐activated chloride currents, were compared with the actions of the calcium channel blocker nifedipine on noradrenaline‐ and 5‐hydroxytryptamine (5‐HT)‐induced pressor responses of the rat perfused isolated mesenteric vascular bed. Bolus injections of noradrenaline (1 and 10 nmol) increased the perfusion pressure in a dose‐dependent manner. Nifedipine (1 μm) inhibited the increase in pressure produced by 1 nmol noradrenaline by 31±5%. Niflumic acid (10 and 30 μm) also inhibited the noradrenaline‐induced increase in perfusion pressure and 30 μm niflumic acid reduced the pressor response to 1 nmol noradrenaline by 34±6%. The increases in perfusion elicited by 5‐HT (0.3 and 3 nmol) were reduced by niflumic acid (10 and 30 μm) in a concentration‐dependent manner and 30 μm niflumic acid inhibited responses to 0.3 and 3 nmol 5‐HT by, respectively, 49±8% and 50±7%. Nifedipine (1 μm) decreased the pressor response to 3 nmol 5‐HT by 44±9%. In the presence of a combination of 30 μm niflumic acid and 1 μm nifedipine the inhibition of the pressor effects of noradrenaline (10 nmol) and 5‐HT (3 nmol) was not significantly greater than with niflumic acid (30 μm) alone. Thus the effects of niflumic acid and nifedipine were not additive. In Ca‐free conditions the transient contractions induced by 5‐HT (3 nmol) were not reduced by 30 μm niflumic acid, suggesting that this agent does not inhibit calcium release from the intracellular store or the binding of 5‐HT to its receptor. Niflumic acid 30 μm did not inhibit the pressor responses induced by KCl (20 and 60 μmol) which were markedly reduced by 1 μm nifedipine. In addition, 1 μm levcromakalim decreased pressor responses produced by 20 μmol KCl. These data suggest that niflumic acid does not block directly calcium channels or activate potassium channels. It is concluded that niflumic acid selectively reduces a component of noradrenaline‐ and 5‐HT‐induced pressor responses by inhibiting a mechanism which leads to the opening of voltage‐gated calcium channels. Our data suggest that the Ca2+‐activated chloride conductance may play a pivotal role in the activation of voltage‐gated calcium channels in agonist‐induced constriction of resistance blood vessels.

The effect of α,β-methylene ATP on the depolarization evoked by noradrenaline (γ-adrenoceptor response) and ATP in the immature rat basilar artery

Depolarizations evoked by noradrenaline that were resistant to α‐ and β‐adrenoceptor antagonists were recorded in the rat basilar artery. These γ‐adrenoceptor‐mediated responses and the depolarizations to adenosine triphosphate (ATP) were blocked by pretreatment of the tissue with α,β‐methylene ATP. These data are discussed with respect to the selectivity of α,β‐methylene ATP.

Membrane mechanism associated with muscarinic receptor activation in single cells freshly dispersed from the rat anococcygeus muscle

1 The mechanism of action of carbachol was studied on freshly dispersed cells of the rat anococcygeus using microelectrodes and patch pipettes. 2 Micro‐ionophoretic application of carbachol evoked reproducible depolarizations which were reduced or blocked by atropine (10−7‐10−6 m). The time courses of the responses to noradrenaline and carbachol were similar. 3 The reversal potential of the carbachol‐induced response was −3.8 mV and similar to the value (‐6.2 mV) found for noradrenaline. 4 During the response to carbachol the membrane conductance was increased. At depolarized membrane potentials carbachol evoked biphasic membrane responses suggesting an increase in two separate ionic conductances. 5 With patch pipettes in the whole‐cell configuration under voltage‐clamp, carbachol produced an inward current at a holding potential of −50 mV. The inward current was associated with an increase in membrane conductance with an equilibrium potential of about 0 mV. 6 It is suggested that muscarinic receptors and adrenoceptors in the rat anococcygeus may activate similar membrane conductances. The most prominent mechanism is an increase in chloride ion conductance.

Electrophysiological analysis of neurogenic vasodilatation in the isolated lingual artery of the rabbit.

1 The nature of neurogenic vasodilatation was investigated in isolated segments of rabbit lingual artery. In separate experiments membrane responses to nerve stimulation were studied by use of microelectrodes. 2 In the presence of guanethidine to block constrictor responses and noradrenaline to induce tone, field stimulation with trains of pulses (8 Hz for 0.5 to 4 s) produced vasodilatation. Atropine (10−6 M) reduced the relaxations to about 50% of the control values while the induced vasodilatations were potentiated by physostigmine. Tetrodotoxin (TTX, 10−7 M) blocked all nerve‐evoked responses. These data suggest that there is a cholinergic and a non‐cholinergic component of the vasodilatation produced by nerve stimulation in the rabbit lingual artery. 3 Single stimuli did not evoke electrophysiological responses. With parameters similar to those used in the mechanical studies, periarterial stimulation in the presence of guanethidine evoked membrane hyperpolarizations which achieved amplitudes of up to 11 mV. The ionophoretic application of acetylcholine (ACh) produced hyperpolarization. 4 The inhibitory junction potentials (i.j.ps) but not the ionophoretic‐induced responses were blocked by TTX. The nerve‐evoked and the ACh‐induced hyperpolarizations were potentiated by physostigmine (5 × 10−7 M) and totally blocked by atropine (10−7 M). 5 I.j.ps and hyperpolarization to ionophoresis of ACh were recorded from arteries in which the endothelium had been removed by mechanical rubbing. Mechanical relaxation to field stimulation and ACh was observed in preparations without endothelium. 6 These data suggest that the cholinergic component of the neurogenic vasodilatation in the rabbit lingual artery is accompanied by hyperpolarization. The non‐cholinergic component does not appear to possess an electrophysiological correlate. In addition, it seems that the action of nerve‐released ACh is mediated by muscarinic receptors which are situated directly on the vascular smooth muscle cells.

Microelectrode study on the ionic mechanisms which contribute to the noradrenaline‐induced depolarization in isolated cells of the rabbit portal vein

1 Experiments were carried out to determine the identity of the ionic mechanisms which contribute to the noradrenaline‐evoked depolarization recorded with microelectrodes in freshly dispersed rabbit portal vein cells. 2 In normal physiological salt solution with microelectrodes containing 1 m NaCl the reversal potential (Er) of the noradrenaline‐induced response was −7.6 ± 2.9 mV. When the external NaCl was replaced by equipmolar concentrations of NaI, NaBr and NaNO3, Er was −33 ± 3.5 mV, −29.1 ± 5.2 mV and −18.4 ± 1.1 mV, respectively. 3 In physiological salt solution Er of noradrenaline‐evoked responses recorded with electrodes filled with 1m NaI or 1m NaNO3 was + 16.3 + 3.9mV and +10.0 ± 7.6 mV, respectively. These results suggest that an increase in anion conductance contributes to the depolarization to noradrenaline. 4 Data from experiments with organic anions indicated that glutamate behaves as a less permeant anion but that benzenesulphonate blocks the anion conductance to unmask another conductance mechanism activated by noradrenaline. 5 When external NaCl was substituted by choline Cl and Tris Cl Er was −21.3 ± 3.7 mV and −20.5 ± 2.8 mV, respectively. These results suggest that noradrenaline also activates a cation conductance mechanism in freshly dispersed rabbit portal vein cells. It is concluded that the depolarization to noradrenaline recorded with a microelectrode is produced by the simultaneous activation of an anion channel and a separate cation channel.

Mechanism of action of α‐adrenoceptor activation in single cells freshly dissociated from the rabbit portal vein

1 The action of noradrenaline was studied in freshly dispersed cells of the rabbit portal vein using microelectrode techniques. 2 In normal physiological salt solution, the ionophoretic application of noradrenaline evoked an α‐adrenoceptor‐mediated depolarization and sometimes a β‐adrenoceptor‐mediated hyperpolarization. Experiments were carried out in the presence of propranolol to study the membrane mechanism associated with α‐adrenoceptor activation. 3 In the current clamp mode of recording, the equilibrium potential of the noradrenaline‐evoked depolarization was −1.9 mV. The depolarization was brought about by an increase in membrane conductance. 4 Under voltage clamp conditions, noradrenaline produced an inward current with a reversal potential of −7 ± 3 mV (mean ± s.e. mean). 5 The relationship between the noradrenaline‐induced inward current and clamp potential was non‐linear. Depolarization enhanced the conductance elicited by noradrenaline. 6 Evidence is presented which suggests that an additional conductance mechanism (probably an increase in potassium conductance) is also evoked by α‐adrenoceptor stimulation in dispersed cells of rabbit portal vein.

Comparison of the biphasic excitatory junction potential with membrane responses to adenosine triphosphate and noradrenaline in the rat anococcygeus muscle.

1 The effects of field stimulation and ionophoretic application of adenosine triphosphate (ATP) and noradrenaline were studied in the rat anococcygeus by means of an intracellular micro‐electrode. 2 Field stimulation at room temperature produced three types of electrical membrane response: (a) a ‘fast’ excitatory junction potential (e.j.p.) which had a latency of less than 100 ms and a time to peak of 300 ms; (b) a ‘slow’ e.j.p. which had a latency of several hundred ms and a time to peak of 1–2 s, and (c) an inhibitory junction potential (i.j.p.) which had a time to peak of about 1.5 s. All three responses were blocked by tetrodotoxin. 3 The ionophoretic application of ATP produced both monophasic and biphasic depolarizations; these responses had a latency of less than 30 ms and a time to peak of 150–300 ms. In contrast, ionophoretically‐applied noradrenaline produced a depolarization which had a mean latency of 471 ms and a time to peak of 861 ms. 4 The ‘slow’ e.j.p. and the noradrenaline‐induced depolarization were blocked by prazosin whereas the ‘fast’ e.j.p. and the ATP responses were resistant to this antagonist and also to atropine. 5 These results are further evidence that the ‘fast’ e.j.p. in some smooth muscle tissues is mediated by ATP.