In the last few years there has been a veritable explosion of knowledge about cyclic nucleotide phosphodiesterases. In particular, the accumulating data showing that there are a large number of different phosphodiesterase isozymes have triggered an equally large increase in interest about these enzymes. At least seven different gene families of cyclic nucleotide phosphodiesterase are currently known to exist in mammalian tissues. Most families contain several distinct genes, and many of these genes are expressed in different tissues as functionally unique alternative splice variants. This article reviews many of the more important aspects about the structure, cellular localization, and regulation of each family of phosphodiesterases. Particular emphasis is placed on new information obtained in the last few years about how differential expression and regulation of individual phosphodiesterase isozymes relate to their function(s) in the body. A substantial discussion of the currently accepted nomenclature is also included. Finally, a brief discussion is included about how the differences among distinct phosphodiesterase isozymes are beginning to be used as the basis for developing therapeutic agents.

Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms

https://www.cell.com/trends/pharmacological-sciences/pdf/0165-6147(88)90238-6.pdf

The pharmacology of potassium channels and their therapeutic potential

A potassium (K) channel that was inhibited by physiological (f1M) concen­ trations of intracellular ATP ([ATP]i) and that opened as [ATP]i decreased was first described in the heart ( l). Subsequently, similar K -channels all with unitary conductances in the range of 40-80 pS (measured under symmetrical high K+ conditions) were also found to exist in insulin-secreting cells and in skeletal muscle (2_5).1 Such channels constitute what are classically described as ATP-sensitive K-channels and were termed Type 1 by Ashcroft & Ashcroft (6). In this review they are designated KATP and the current flowing through them is defined as IK(ATP) . Type 1 KATP channels are essentially calciumand voltage-independent, K+ -selective, and are half-maximally inhibited by [ATP]i in the range 10-100 fJ.M. They exhibit inward rectification and a key pharmacological feature is their inhibition by agents like tolbutamide and glyburide . Other ATP-sensitive K-channels exist and these were designated Types 2, 3, 4, and 5 (6). Such channels vary not only in their sensitivity to calcium and to the inhibitory effects of [ATP]i, but also in their selectivity for potassium and their susceptibility to pharmacological modulation.

The Pharmacology of ATP-Sensitive Potassium Channels

Physiological roles for adenosine and adenosine 5'-triphosphate in the nervous system.

1. Whole-cell membrane current recordings under voltage clamp were made at room temperature from dispersed single cells of longitudinal smooth muscle of rabbit jejunum and dispersed single smooth muscle cells of rabbit ear artery using patch pipettes containing up to 10 mM-EGTA Ca buffer. 2. Spontaneous transient outward currents (s.t.o.c.s) up to 250 pA in size and about 100 ms in duration were observed in conditions which might lead to an elevated internal Ca concentration. The amplitude distribution in some cells and form of the currents suggested that they were evoked by a quantal stimulus. 3. S.t.o.c. amplitude was voltage dependent and reversed at the K equilibrium potential. S.t.o.c.s. were blocked by 1 mM-tetraethylammonium or 10 mM-Ba applied externally or by perfusing Cs inside the cell. 4. Removing external Ca abolished s.t.o.c. activity in the jejunal cells but not in arterial cells. Increasing EGTA buffering within the cells from 1 mM or less to 10 mM abolished activity in both cell types. 5. Caffeine (5 mM) applied to the bathing solution stimulated rapid discharge of transient outward currents and then a prolonged period of inhibition. The stimulated discharge was sensitive to external Ca in jejunal cells but much less so in arterial cells. ACh applied by ionophoresis to jejunal cells or noradrenaline bath-applied onto arterial cells also stimulated discharge of transients followed by a prolonged inhibitory phase. 6. It was suggested that s.t.o.c.s represent the simultaneous opening of up to 75-100 Ca-activated K channels at -40 mV in response to sudden discharge of Ca from internal stores when these became overloaded, and that this process may occur cyclically.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1986.sp016333

Spontaneous transient outward currents in single visceral and vascular smooth muscle cells of the rabbit.

BRL34915 (0.1-10 microM) suppressed the spontaneous tone of guinea-pig isolated trachealis in a concentration-dependent manner. BRL34915 was not antagonized by propranolol (1 microM). In trachea where spontaneous tone was suppressed by indomethacin (2.8 microM) but subsequently restored to the same level with acetylcholine or histamine, the relaxant potency of BRL34915 was reduced. In Krebs solution containing K+ (120 mM), isolated trachealis muscle developed near-maximal tension. The relaxant effects of BRL34915 were virtually abolished in this medium. Concentration-effect curves for KCl, acetylcholine and histamine were constructed in tissues treated with indomethacin (2.8 microM). BRL34915 (10 microM) depressed the foot of the concentration-effect curve for KCl and caused minor rightward shifts in the concentration-effect curves of acetylcholine and histamine. Four K+-channel inhibitors were tested. Apamin (0.1 microM) did not modify the action of BRL34915. Tetraethylammonium (8 mM) had little effect but procaine (5 mM) and 4-aminopyridine (5 mM) each significantly inhibited the relaxant action of BRL34915. Intracellular electrophysiological recording showed that BRL34915 (0.1 microM) caused very minor relaxation and little, if any, electrical change. Higher concentrations (1-10 microM) evoked relaxation, suppression of spontaneous electrical slow waves and marked hyperpolarization of the trachealis cells. In the presence of TEA (8 mM) or procaine (5 mM) the hyperpolarization induced by BRL34915 was significantly reduced. In trachealis skinned of its plasma membranes, tension development induced by Ca2+ (20 microM) was unaffected either by BRL34915 (10 microM) or by nicorandil (1 mM). In studies of the efflux of 86Rb+ from muscle-rich strips of trachea, BRL34915 (1 and 10 microM) increased the efflux rate constant. It is concluded that BRL34915 evokes relaxation of the trachealis by a mechanism that involves neither beta-adrenoceptor activation nor direct reduction of the sensitivity of the intracellular contractile machinery to cytosolic free Ca2+. The action of BRL34915 may depend on the opening of K+ channels in the plasma membrane which are permeable to 86Rb+. The opening of these channels, or the effects of their opening, may be reduced by K+-channel inhibitors such as 4-aminopyridine, procaine and TEA but not by apamin.

/pdf/electrical-and-mechanical-effects-of-brl34915-in-guinea-pig-1neu52dzf3.pdf

Electrical and mechanical effects of BRL34915 in guinea‐pig isolated trachealis

The effects of pinacidil have been compared with those of glyceryl trinitrate (GTN) using the aorta and portal vein of the rat and the trachealis and taenia caeci of the guinea-pig. In aorta, both pinacidil and GTN inhibited responses to noradrenaline and showed some selective inhibition of contractions to 20 mM K+. Responses to 80 mM K+ were little affected. In trachealis, both pinacidil and GTN inhibited spontaneous tone and selectively relaxed spasms to 20 mM K+. Responses to 80 mM K+ were unaffected. In portal vein, pinacidil completely inhibited spontaneous electrical and mechanical activity. GTN reduced the amplitude of tension waves and extracellularly-recorded discharges, but increased the frequency of spontaneous electrical and mechanical activity. In portal vein, pinacidil inhibited contractions to noradrenaline and selectively inhibited responses to 20 mM K+. GTN had little inhibitory effect on responses to either noradrenaline or K+. In portal veins loaded with 86Rb as a K+-marker, pinacidil significantly increased the 86Rb efflux rate coefficient whilst GTN had no effect on 86Rb exchange. In taenia caeci, both pinacidil and GTN inhibited the spontaneous tone of the preparation. These inhibitory effects were not antagonized by apamin. It is concluded that pinacidil and GTN do not share a common relaxant mechanism. Evidence has been obtained that pinacidil exerts its inhibitory effects by the opening of apamin-insensitive, 86Rb-permeable K+ channels.

Evidence that the mechanism of the inhibitory action of pinacidil in rat and guinea-pig smooth muscle differs from that of glyceryl trinitrate.

1
In trachealis bathed by a K+-rich, Ca2+-free physiological salt solution, calcium chloride (CaCl2) at 0.01 to 10 mmol l−1 evoked concentration-dependent spasm. Verapamil (0.1 to 10 μmol l−1) was an effective antagonist of CaCl2.

2
Spasm evoked by acetylcholine, histamine, potassium chloride (KCl) and tetraethylammonium (TEA) was studied in trachealis bathed by normal Krebs solution. Verapamil (0.1 to 10 μmol l−1) markedly suppressed spasm evoked by KCl and TEA. In contrast the actions of acetylcholine and histamine were much less affected by verapamil.

3
Spasm evoked by prostaglandin E2 was studied in trachealis bathed by Krebs solution containing indomethacin (2.8 μmol l−1). Verapamil (0.1 to 10 μmol l−1) had little or no effect against prostaglandin E2-induced spasm.

4
Verapamil (0.1 to 10 μmol l−1) had relatively little effect on the tone of trachealis bathed by normal Krebs solution. In contrast bathing in Krebs solution lacking CaCl2 caused almost complete tone loss.

5
Extracellular electrophysiological recording showed that verapamil (10 μmol l−1) suppressed not only TEA-evoked spasm but also TEA-evoked slow waves and spike potentials. Verapamil also abolished the transient period of slow wave activity associated with the spasm evoked by KCl.

6
Intracellular electrophysiological recording showed that TEA-induced spike activity was resistant to tetrodotoxin (3 μmol l−1). However, verapamil (10 μmol l−1) abolished the tetrodotoxin-resistant spikes without increasing the resting membrane potential.

7
It is concluded that verapamil suppresses TEA- or KCl-induced spasm, slow waves or spikes by inhibition of Ca2+ influx. Spasm evoked by acetylcholine, histamine and prostaglandin E2 depends on mechanisms for increasing the cytoplasmic concentration of free Ca2+ which are resistant to verapamil. The failure of verapamil markedly to depress tissue tone is consistent with the proposal that tone results from the activity of endogenous prostaglandins.

Antagonism of Ca2+ and other actions of verapamil in guinea‐pig isolated trachealis

1 BRL 34915 (0.04-1.3 microM) caused concentration-dependent inhibition of spontaneous phasic spasms of the isolated uterus of the term pregnant rat and this effect was not antagonized by propranolol. Spasms evoked by low concentrations of KCl (less than 20 mM) were inhibited by BRL 34915 but those evoked by higher concentrations (greater than 40 mM) were unaffected. 2 In experiments using extracellular electrical recording, BRL 34915 (10 microM) selectively inhibited oxytocin-induced phasic spasms and the associated spike activity but had little effect on the tonic component of the spasms. BRL 34915, as an inhibitor of phasic spasms to oxytocin (0.2 nM), was antagonized by procaine (0.3 and 1 mM). 3 BRL 34915 (10 microM) did not inhibit Ca2+-induced spasm of saponin-skinned thin myometrial strips. 4 Intracellular microelectrode recording from myometrial strips showed that BRL 34915 (10 microM) inhibited action potentials and phasic spasms in the presence of oxytocin (0.2 nM) and produced a hyperpolarization of 5 mV. 5 In single myometrial cells under current or voltage clamp, BRL 34915 (10 microM) had no effect on action potentials and inward current in Ca2+- or Ba2+-containing media in the presence of tetraethylammonium, 4-aminopyridine and caesium chloride. In the absence of these K+-channel inhibitors, BRL 34915 had no effect on resting membrane potential, membrane resistance, action potential, inward current or outward current. 6 BRL 34915 (1 or 10 microM) had no effect on 86Rb efflux from myometrial strips. 86Rb efflux was increased by oxytocin (0.2 and 20 nM). 7 The relaxant profile of BRL 34915 in the rat uterus is similar to that described for other smooth muscles where an action to open membrane K+-channels has been proposed. BRL 34915 inhibited spike production but produced only a small hyperpolarization without a detectable increase in 86Rb efflux. Membrane resistance and transmembrane currents were unaffected. These results suggest that in the uterus the effects of BRL 34915 may be restricted to K+-channels involved in the production of pacemaker activity.

/pdf/the-relaxant-action-of-brl-34915-in-rat-uterus-4oddmx7t6w.pdf

The relaxant action of BRL 34915 in rat uterus.

1 Tetraethylammonium (TEA, 1-8 mmol/l) evoked spasm of guinea-pig trachealis which was unaffected by atropine (1 mumol/l), mepyramine (1 mumol/l) or tetrodotoxin (3 mumol/l). 2 The spasm evoked by TEA was markedly suppressed in Ca2+-free Krebs solution while that evoked by acetylcholine was much less affected. 3 Extracellular electrical recording showed that exposure to Ca2+-free Krebs solution suppressed both spontaneous electrical slow wave activity of the trachea and the spasm and slow waves induced by TEA. These effects were reversible. 4 TEA (2 and 8 mmol/l) increased the lanthanum-resistant calcium fraction of trachea. 5 It is concluded that TEA acts directly on the smooth muscle of guinea-pig trachea, that the spasm and electrical slow waves evoked are Ca2+-dependent and that the cellular influx of Ca2+ is increased.

/pdf/evidence-that-the-spasmogenic-action-of-tetraethylammonium-gnbs8qculw.pdf

Evidence that the spasmogenic action of tetraethylammonium in guinea-pig trachealis is both direct and dependent on the cellular influx of calcium ion.

Roger C. Small

Papers

Electrical and mechanical effects of BRL34915 in guinea‐pig isolated trachealis

Evidence that the mechanism of the inhibitory action of pinacidil in rat and guinea-pig smooth muscle differs from that of glyceryl trinitrate.

Antagonism of Ca2+ and other actions of verapamil in guinea‐pig isolated trachealis

The relaxant action of BRL 34915 in rat uterus.

Evidence that the spasmogenic action of tetraethylammonium in guinea-pig trachealis is both direct and dependent on the cellular influx of calcium ion.