Studies on new phosphodiesterase inhibitors. I. Synthesis of 1-(2,3-epoxypropoxy)-2(4)-fluorobenzenes and 1-(2-hydroxy-3-morpholinopropoxy and piperazino)fluorobenzene derivatives.

A series of 1-(2,3-epoxypropoxy)-2(4)-fluorobenzenes and their corresponding 1-(2-hydroxy-3-morpholinopropoxy and piperazino)fluorobenzene derivatives (8a--f) were synthesised via a short synthetic route in good chemical yields and were evaluated for inotropic and chronotropic activity in isolated guinea-pig atria preparation. The compounds act as potential phosphodiesterase (PDE) inhibitors with desirable biological activity and have considerable promise in heart therapy.

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)

Membrane ionic mechanisms activated by noradrenaline in cells isolated from the rabbit portal vein.

1. Membrane currents were recorded in cells freshly dispersed from the rabbit portal vein with patch pipette techniques in the whole-cell configuration of recording. In potassium-containing solutions at a holding potential of -50 mV noradrenaline usually evoked an inward current and enhanced greatly the outward current evoked by depolarizing voltage steps. 2. The ionic mechanism of the inward current was investigated in potassium-free solutions in which the inward current elicited by noradrenaline was produced by an increase in membrane conductance. 3. In the first series of experiments NaCl was the main salt in the patch pipette solution (representing the intracellular milieu) and the ionic composition of the bathing solution was altered. In these conditions the reversal potential of the noradrenaline-induced current was close to the chloride equilibrium potential (ECl). 4. When sodium glutamate was the major salt in the pipette solution the reversal potential of the noradrenaline-evoked current was influenced by the cation rather than the anion gradient. 5. With 89 mM-BaCl2 in the external solution (and sodium glutamate in the pipette) noradrenaline produced a membrane current with a reversal potential which was much more positive than ECl or ENa. These results indicate that noradrenaline opens a chloride-selective channel and a cation channel which is permeable to monovalent and divalent cations. 6. Bath application of 10 mM-caffeine evoked a membrane current with a reversal potential close to ECl with either NaCl or sodium glutamate in the pipette. This is interpreted to mean that the increase in membrane chloride conductance can occur as a consequence of a rise in intracellular calcium concentration. It is less evident that the cation channel is calcium activated.

Mechanism of action of alpha-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 alpha-adrenoceptor-mediated depolarization and sometimes a beta-adrenoceptor-mediated hyperpolarization. Experiments were carried out in the presence of propranolol to study the membrane mechanism associated with alpha-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 alpha-adrenoceptor stimulation in dispersed cells of rabbit portal vein.

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.

Action of noradrenaline on single smooth muscle cells freshly dispersed from the rat anococcygeus muscle.

1. Membrane responses were recorded using a standard patch pipette technique in isolated cells freshly dispersed from the rat anococcygeus muscle. Using the whole-cell mode of recording, the ionophoretic application of noradrenaline evoked depolarization (current clamp) and inward current (voltage clamp) at membrane potentials of -50 to -60 mV. 2. The inward current to noradrenaline was reversibly blocked by low concentrations of phentolamine indicating that the response is mediated by alpha-adrenoceptors. The inward current was associated with a conductance increase. 3. The ionic mechanism of the inward current was investigated in potassium-free solutions. Alteration of the chloride equilibrium potential produced similar changes in the reversal potential of the noradrenaline-induced current and it is concluded that noradrenaline opens chloride-selective channels. 4. The noradrenaline-activated chord conductance was increased by depolarization. From a holding potential of -50 mV depolarizing voltage steps in the presence of noradrenaline produced relaxations and the time constants of these relaxations at -50 and +30 mV were respectively 89 and 166 ms. 5. The calcium ionophore A23187 and caffeine applied in the bathing solution also induced an increase in chloride conductance. 6. It is concluded that in isolated cells of the rat anococcygeus muscle the depolarization to noradrenaline is mediated by an increase in membrane chloride conductance.

Action of externally applied adenosine triphosphate on single smooth muscle cells dispersed from rabbit ear artery.

1. Adenosine triphosphate (ATP), applied in the bathing solution or ionophoretically, depolarized freshly dispersed single arterial smooth muscle cells obtained by collagenase and elastase treatment of the rabbit ear artery. 2. Ionophoretic application of ATP evoked an inward current with a latency of about 70 ms and a time to peak of about 230 ms in cells held under voltage clamp using whole-cell patch-pipette techniques. 3. Bath application of 10 microM-ATP evoked a transient inward current at negative holding potentials. The amplitude of the ATP-induced current was linearly related to the clamp potential with a reversal potential near 0 mV. Removal of extracellular calcium, buffering intracellular calcium with high EGTA concentration, or depleting calcium stores with caffeine or noradrenaline treatment did not affect the ATP-evoked current. 4. Changing the chloride concentration gradient by decreasing extracellular or intracellular chloride concentration, or using the chloride channel blocker, frusemide, had no effect on the currents. 5. Replacing sodium with Tris shifted the reversal potential to more negative potentials. The reversal potential was not affected by exchanging intracellular potassium for caesium or sodium. Replacing extracellular sodium with 89 mM-barium also had little effect on the reversal potential. 6. These results are consistent with ATP activating a conductance that is cation selective but allows both monovalent and divalent cations to pass across the membrane.

The action of noradrenaline on single smooth muscle cells freshly dispersed from the guinea-pig pulmonary artery.

Responses to ionophoretically-applied noradrenaline were investigated with micro-electrodes in whole tissue preparations and with patch pipettes in isolated cells dispersed from the guinea-pig pulmonary artery. In whole tissue and dispersed cells noradrenaline evoked monophasic depolarizations which had a similar time course. In dispersed cells the amplitude of electronic potentials was reduced during the noradrenaline-evoked depolarization. Under voltage clamp noradrenaline elicited an inward current, which persisted in 18 mM external potassium with the membrane potential set at the potassium equilibrium potential. In voltage clamp experiments the amplitude of current steps to hyperpolarizing voltage jumps was increased during the noradrenaline-induced inward current. These data suggest that the depolarization to noradrenaline in the guinea-pig pulmonary artery is mediated by an increase in membrane conductance.

The effect of alpha, beta-methylene ATP on the depolarization evoked by noradrenaline (gamma-adrenoceptor response) and ATP in the immature rat basilar artery.

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

Evidence for two mechanisms of depolarization associated with alpha 1-adrenoceptor activation in the rat anococcygeus muscle.

Membrane potential responses in the rat isolated anococcygeus to bath-applied noradrenaline and field stimulation have been investigated by use of intracellular microelectrode and combined extracellular electrical and mechanical (sucrose gap) recording techniques. Intracellular recordings were made usually from tissues immobilized with hypertonic Krebs solution. Bath-application of noradrenaline produced depolarizations which consisted of two components; an initial 'fast' phase which peaked within 1-2 s and which was followed by a 'slow' sustained depolarization. Both components were concentration-dependent. Noradrenaline could also evoke oscillations in membrane potential which, unlike the 'fast' component of depolarization, were prevented by conditioning hyperpolarization of the membrane and were evoked by direct membrane depolarization with externally applied current pulses. Thus, the oscillations are voltage-dependent phenomena. Replacement of the external NaCl of the Krebs solution with an equimolar amount of Na benzenesulphonate abolished the noradrenaline-evoked 'fast' depolarization while the 'slow' phase was unaffected. This suggests that two mechanisms of depolarization are activated in this muscle by the bath-application of noradrenaline. The adrenergic excitatory junction potential was also abolished in Na benzenesulphonate. Prazosin reduced both the 'fast' and 'slow' components of depolarization produced by noradrenaline indicating their mediation by alpha 1-adrenoceptors. The membrane potential (-29 mV) at the maximum amplitude of the 'fast' depolarization was similar to the equilibrium potential (-27 mV) for the depolarization evoked by ionophoretically applied noradrenaline and which was obtained by extrapolation from the relationship between amplitude of the ionophoretic response and membrane potential displacement in the partition chamber. These results suggest that the 'fast' depolarization and the ionophoretic response are due to an increased membrane conductance, possibly to chloride.

Electrophysiological analysis of the nature of adrenoceptors in the rat basilar artery during development.

The nature of adrenoceptors in basilar arteries of neonatal rats was investigated by means of electrophysiological techniques. In immature (2-6 day postnatal) rats, micro-injection of noradrenaline elicited a depolarization which consisted of two components. The initial 'fast' component (time to peak of 0.3-4s) was slightly reduced by phentolamine and was not antagonized by propranolol. The second 'slow' component (time to peak of about 50s) was not blocked by phentolamine but was antagonized by low concentrations (10(-7) M) of propranolol. In immature rats, micro-injection of isoprenaline was more potent than noradrenaline in evoking the 'slow' depolarization but less effective in eliciting the 'fast' response. The pharmacology with respect to adrenoceptor antagonists of both components of the isoprenaline- and noradrenaline-induced depolarizations was similar. There was some evidence of inhibitory beta-adrenoceptors in immature rat basilar vessels. In adult rats (6 week old) noradrenaline produced a large 'fast' depolarization which was followed by a 'slow' tail response. Both components were not antagonized by phentolamine or propranolol. It appears that in the basilar artery of neonatal rats there are excitatory alpha- and inhibitory beta-adrenoceptors but the major responses to noradrenaline and isoprenaline are mediated by gamma- and excitatory beta-receptors. In adult animals the gamma-adrenoceptor predominates. Experiments were carried out in which agonists were applied by ionophoresis. These results confirm the presence of excitatory beta-receptors in neonatal basilar vessels and show the response has slow kinetics and it is likely that the beta-receptors are distributed uniformly over the smooth muscle surface. In adult animals it was not possible to elicit an excitatory beta-receptor-mediated response. The ionophoretic application of noradrenaline never evoked a perceptible depolarization which could be attributed to gamma-adrenoceptor stimulation. This result is discussed in terms of receptor distribution with respect to synaptic function in a syncytium.

Mechanisms underlying electrical and mechanical responses of the bovine retractor penis to inhibitory nerve stimulation and to an inhibitory extract.

The response of the bovine retractor penis (BRP) to stimulation of non-adrenergic, non-cholinergic (NANC) inhibitory nerves and to an inhibitory extract prepared from this muscle have been studied using intracellular microelectrode, sucrose gap and conventional mechanical recording techniques. Both inhibitory nerve stimulation and inhibitory extract hyperpolarized the membrane potential and relaxed spontaneous or guanethidine (3 X 10(-5) M)-induced tone. These effects were accompanied by an increase in membrane resistance. Following membrane potential displacement from an average value of -53 +/- 7 mV (n = 184; Byrne & Muir, 1984) inhibitory potentials to nerve stimulation were abolished at approximately -30 mV; there was no evidence of reversal. Displacement by inward hyperpolarizing current over the range -45 to -60 mV increased the inhibitory response to nerve stimulation and to inhibitory extract; at more negative potential values (above approximately -60 mV) the inhibitory potential decreased and was abolished (approximately -103 mV). There was no evidence of reversal. Removal of [K+]o reversibly reduced hyperpolarization to nerve stimulation and inhibitory extract. No enhancement was observed. Increasing the [K+]o to 20 mM reduced the inhibitory potential to nerve stimulation but this was restored by passive membrane hyperpolarization. Inhibitory potentials were obtained at membrane potential values exceeding that of the estimated EK (-49 mV). [Cl-]o-free or [Cl-]o-deficient solutions reduced and abolished (after some 20-25 min) the hyperpolarization produced by inhibitory nerve stimulation or inhibitory extract. The inhibitory potential amplitude following nerve stimulation was not restored by passive displacement of the membrane potential from -26 to -104 mV approximately. Ouabain (1-5 X 10(-5) M) reduced then (45-60 min later) abolished the inhibitory potential to nerve stimulation. The effects of this drug on the extract were not investigated. It is concluded that the inhibitory response to nerve stimulation and extract in the BRP may involve several ionic species. However, unlike that in gastrointestinal muscles the NANC response in the BRP is accompanied by an increased membrane resistance and does not primarily involve K+. The underlying mechanisms for the inhibitory response to both NANC nerve stimulation and inhibitory extract appear to be similar, compatible with the view that the latter may contain the inhibitory transmitter released from these nerves in this tissue.

Electrical and mechanical responses of the bovine retractor penis to nerve stimulation and to drugs.

The responses of the bovine retractor penis muscle to field stimulation of intramural nerves in vitro was investigated using micro-electrode and extracellular (sucrose gap) techniques. In the absence of tone single pulses or trains of stimuli (1-50 Hz 0.1-0.5 m.sec) produced e.j.p.s and a decrease in membrane resistance; spike potentials were not observed. E.j.p.s often small in amplitude (3-5 mV to single pulse) and accompanied by contractions in almost all preparations were noradrenergic, abolished by guanethidine (1-3 x 10(-5) M) and tetrodotoxin (3.5 x 10(-6) M) but not by prazosin (0.05 - 1.4 x 10(-6) M). Prazosin abolished the depolarization and contraction produced by added NA (0.02 - 2 x 10(-8) moles). TEA (10(-2) M) depolarized the membrane and initiated spontaneous activity; e.j.p.s and contractions were enhanced and prolonged but no spikes were observed. Atropine (0.5 x 10(-6) M) increased and physostigimine (1-5 x 10(-6) M) decreased e.j.p.s and contractions indicating a cholinergic regulatory component in the release of the excitatory transmitter. In the presence of tone, nerve stimulation produced i.j.p.s. and relaxations which were unaffected by apamin (5 x 10(-7) M), atropine (3 x 10(-6) M), guanethidine (3 x 10(-5) M), phentolamine (5 x 10(-6) M) and propranolol (4 x 10(-6) M) but were abolished by tetrodotoxin (3.5 x 10(-7) M) suggesting their mediation by non-adrenergic non-cholinergic (NANC) nerves. Sodium nitroprusside (10(-10) - 10(-8) moles), which increases cyclic GMP, also hyperpolarized the membrane and relaxed the BRP. Those responses and those to inhibitory nerve stimulation were antagonized by oxyhaemoglobin which inhibits guanylate cyclase. 2-O-propoxyphenyl-8-azapurin-6-one (M & B 22948 3-9 x 10(-6) M) which inhibits cGMP-specific phosphodiesterase, enhanced the relaxation but not the i.j.p. TEA (10(-2) M) initially depolarized the membrane potential and raised tone. In the sucrose gap inhibitory potentials were abolished; the mechanical relaxation was not and a small contractile component emerged. Electrical and mechanical inhibitory components in the bovine retractor penis may not be correlated.

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

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 microelectrode. 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. 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. 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. These results are further evidence that the 'fast' e.j.p. in some smooth muscle tissues is mediated by ATP.