Jacob Krier

ENDOTHELIN MODULATES CALCIUM CHANNEL CURRENT IN NEURONES OF RABBIT PELVIC PARASYMPATHETIC GANGLIA

1 The effects of endothelin were studied, in vitro, on neurones contained in the rabbit vesical pelvic ganglion by use of intracellular and single‐electrode voltage clamp techniques under conditions where sodium and potassium channels were blocked. 2 In the current‐clamp experiments, endothelin (1 μm) caused a depolarization followed by a hyperpolarization of the membrane potential. In the voltage‐clamp experiments, endothelin (0.01–1 μm) caused an inward current followed by an outward current in a concentration‐dependent manner. 3 Membrane conductance was increased during the endothelin‐induced depolarization and inward current. Membrane conductance was decreased during the endothelin‐induced hyperpolarization and outward current. 4 The endothelin‐induced inward and outward currents were not altered by lowering external sodium concentration or raising external potassium concentration. 5 The endothelin‐induced inward current was depressed (mean 72%) in a Krebs solution containing nominally zero calcium and high magnesium. These results suggest that a predominent component of the endothelin‐induced inward current is mediated by calcium ions. 6 The calcium‐insensitive component of the inward current was abolished by a chloride channel blocker, 4‐acetamide‐4′‐isothiocyanostilbene‐2,2′‐disulphonic acid. The mean reversal potential for the calcium‐insensitive component of the inward current was −18 mV. This value is near the equilibrium potential for chloride. Thus, it is presumed that the calcium‐insensitive component of the inward current is carried by chloride ions. 7 Endothelin caused an initial depression followed by a long lasting facilitation of both rapidly and slowly decaying components of high‐threshold calcium channel currents (N‐ and L‐type). 8 In summary, the data show that for neurones in the vesical pelvic ganglia, endothelin causes membrane depolarization and activates an inward current. The ionic mechanisms involve receptor‐operated calcium and chloride currents. Also, endothelin causes an initial depression followed by a long‐lasting facilitation of the voltage‐dependent calcium current.

Stimulation of lumbar sympathetic nerves evokes contractions of cat colon circular muscle mediated by ATP and noradrenaline.

1 The action of the lumbar sympathetic nerves to cat colon was studied in vitro using isolated muscle strips with attached lumbar colonic nerves (LCN) orientated in the axis of circular muscle layer. Electrical stimulation of LCN caused frequency‐dependent increases in resting tension and in amplitude of spontaneous contractions. Contractile responses were abolished by tetrodotoxin (3 μm) and by guanethidine (30 μm), indicating that they were neurogenic, involving the release of neurotransmitter from sympathetic fibres. 2 Propranolol (1–9 μm), a β‐adrenoceptor antagonist, caused a concentration‐dependent potentiation of LCN‐evoked contractile responses. Propranolol (3 μm) potentiated contractile responses to exogenously applied noradrenaline but not to phenylephrine. 3 Phentolamine (1–9 μm), an α‐adrenoceptor antagonist, and prazosin (1–9 μm), an α1‐adrenoceptor antagonist, caused a concentration‐dependent reduction of amplitude but did not abolish LCN‐evoked contractile responses. Prazosin (3 μm) or phentolamine (3 μm) antagonized contractile responses to noradrenaline and phenylephrine. 4 Desensitization of purinoceptors with the P2x‐receptor agonist, α,β‐methylene ATP, caused a decrease in amplitude of LCN‐evoked contractile responses and abolished contractile responses to ATP. In muscle strips where α1‐adrenoceptors were blocked with prazosin (3 μm) and P2‐purinoceptors were desensitized with α,β‐methylene ATP, the amplitude of contractile responses was reduced by 82–100%. 5 The P2X‐purinoceptor antagonists, arylazido amino propyl adenosine triphosphate (ANAPP3) and suramin, affected LCN‐evoked contractile responses. ANAPP3 (50–100 μm) caused a concentration‐dependent reduction in the amplitude of contractile response. Suramin (100 μm) caused a small reduction in amplitude of contractile responses but potentiated their amplitude at a concentration of 500 μm. 6 ANAPP3 (100 μm) irreversibly inhibited contractions to α,β‐methylene ATP or ATP. Suramin (100–500 μm) inhibited contractions to α,β‐methylene ATP (0.5–1 μm) or low concentrations of ATP (10–50 μm) but potentiated contractions at higher concentrations. ANAPP3 (100 μm) and suramin (100, 500 μm) had no affect on contractile responses to noradrenaline. 7 Clonidine (0.05–1 μm), a selective α2‐adrenoceptor agonist, caused a concentration‐dependent reduction in amplitude of LCN‐evoked contractile responses, at 10 Hz, while yohimbine (0.1–1 μm), a selective α2‐adrenoceptor antagonist, increased them. At 1 μm, both compounds affected LCN‐evoked contractions at all frequencies. This suggests that prejunctional α2‐receptors are involved in autoinhibition at sympathetic terminals. 8 In summary, LCN‐evoked contractile responses involve the corelease of noradrenaline and ATP or a related purine nucleotide from sympathetic fibres. It is likely that the neurogenic responses are mediated through excitatory postjunctional α1‐adrenoceptors, excitatory suramin‐sensitive and suramin‐insensitive P2X‐purinoceptors and inhibitory β‐adrenoceptors. Also, autoinhibitory prejunctional α2‐adrenoceptors regulate the LCN excitatory pathway to cat colon circular muscle.

δ-Opioid receptors mediate inhibition of fast excitatory postsynaptic potentials in cat parasympathetic colonic ganglia

1 The effects of opioids on synaptic transmission in cat sacral parasympathetic colonic ganglia were studied in vitro, using intracellular electrophysiological techniques. Electrical stimulation of the pelvic nerve evoked fast excitatory postsynaptic potentials (e.p.s.ps), which were blocked by hexamethonium and tetrodotoxin. 2 [d‐Pen2, d‐Pen5] enkephalin and [Met5]enkephalinamide, δ‐opioid receptor agonists, caused concentration‐dependent, reversible depression of fast e.p.s.ps, but had no effect on depolarizations evoked by pressure ejection of the nicotinic agonist 1,1‐dimethyl‐4‐phenyl‐piperazinium. Cell transmembrane potential and membrane input resistance were also unaffected. 3 U‐50,488H, a κ‐opioid receptor agonist, had a very small depressant action while [d‐Ala2, MePhe4, Gly‐ol5] enkephalin, a μ‐opioid receptor agonist, had no effect on fast e.p.s.p. amplitude. Neither compound affected cell transmembrane potential or membrane input resistance. 4 The inhibitory actions of [d‐Pen2, d‐Pen5] enkephalin were antagonized by both naloxone, an antagonist at each of the three opioid receptor types, and by ICI 174,864, an antagonist selective for δ‐opioid receptors. 5 Naloxone and ICI 174,864 both also potentiated fast e.p.s.p. amplitude per se in 50% of cells tested. 6 It is concluded that exogenous opioids act at presynaptic δ‐opioid receptors to inhibit sacral parasympathetic synaptic transmission in cat colonic ganglia in vitro. Furthermore, the effects of opioid anatagonists alone, suggest that endogenous opioids may also be released by preganglionic nerve stimulation and so regulate the release of acetylcholine in these ganglia.

Action potentials and membrane currents of isolated single smooth muscle cells of cat and rabbit colon

Membrane potentials, action potentials and macroscopic currents in enzymatically dispersed, single smooth muscle cells of the circular layer of cat and rabbit colon were investigated. The cells did not exhibit spontaneous depolarizations and repolarizations (slow waves) or spontaneous action potentials. Single action potentials of smooth muscle cells were evoked by depolarizing current pulses of 5 ms to 3 s duration. A repetitive action potential discharge and an increase in the duration of the action potential was observed in cells during long depolarizing current pulses by superfusion with tetraethylammonium (TEA) or 4-aminopyridine (4-AP). Tetrodotoxin (TTX) did not alter the configuration of the action potential. Voltage-clamp experiments revealed two major outward macroscopic currents: a quasi-instantaneous (time-independent) and a time-dependent outward current. Both currents were identified as potassium (K) currents due to their pharmacological sensitivity to K antagonists [TEA, 4-AP and cesium (Cs)] and due to the reversal potential of outward tail currents. Barium selectively blocked the time-independent current. A time-dependent outward K current in colon cells was observed which appeared to be dependent upon entry of calcium ions (Ca2+) through voltage-dependent Ca-channels, since it was blocked by cadmium and low concentrations of nifedipine. The majority of cells did not exhibit transient outward currents. Inward currents were exposed in some of the cells when the K currents were blocked by external TEA and by replacement of K by Cs and TEA in the recording pipette. Inward currents were presumably carried by Ca2+, since they were not altered by TTX, were sensitive to external Ca concentrations and were abolished by the Ca channel antagonist, nifedipine. Carbachol augmented the amplitude of the inward Ca current.

Contractions mediated by α1-adrenoceptors and P2-purinoceptors in a cat colon circular muscle

1 The postjunctional excitatory and inhibitory effects of adrenoceptor and purinoceptor agonists and antagonists were studied in circular smooth muscle strips of cat colon. 2 In the presence of tetrodotoxin (0.5 μm), noradrenaline caused contraction or relaxation of circular smooth muscle at resting tension or with raised tone, respectively. The noradrenaline‐evoked contractions were potentiated and the noradrenaline‐evoked relaxations were antagonized by propranolol (1 μm), suggesting β‐adrenoceptor involvement. 3 At resting tension, noradrenaline, adrenaline and the selective α1‐adrenoceptor agonist, phenylephrine, caused concentration‐dependent contractile responses, with EC50 values of 1.8 ± 0.2 μm, 1.9 ± 0.4 μm and 4.3 ± 1.7 μm, respectively. The EC50 values and the amplitude of maximal responses were not significantly different from one another. Clonidine (0.1–500 μm), a selective α2‐adrenoceptor agonist, was not effective. 4 Prazosin (0.1–9 μm), competitively antagonized the contractile effects of noradrenaline with an estimated pA2 value of 6.93 and a slope of 1.07 ± 0.03. The Kb values, estimated from a single shift (0.1 μm prazosin) of the concentration‐response curves to noradrenaline, adrenaline and phenylephrine were 92.8 ± 9.3 nM, 108.7 ± 6.4 nM and 18.4 ± 3.1 nM, respectively. 5 At resting tension, adenosine 5′ triphosphate (ATP, 5–1000 μm), α,β‐methylene adenosine 5′‐triphosphate (α,β‐MeATP, 0.05–50 μm), β,γ‐methylene adenosine 5′‐triphosphate (β,γ‐MeATP, 0.5–100 μm), and 2‐methylthioadenosine 5′‐triphosphate (2‐MeSATP, 1–500 μm) caused concentration‐dependent contractions with EC50 values of 60.5 ± 15.9 μm, 0.7 ± 0.1 μm, 7.6 ± 0.1 μm and 25.3 ± 12.8 μm, respectively. The maximal responses to α,β‐MeATP and β,γ‐MeATP were greater than maximal responses to 2‐MeSATP and ATP. 6 Suramin (50–500 μm), competitively antagonized the contractile responses of α,β‐MeATP with an estimated pA2 value of 4.92 and a slope of 1.08 ± 0.04. The Kb values, estimated from a single shift (100 μm suramin) of the concentration‐response curves to ATP, α,β‐MeATP, β,γ‐MeATP and 2MeSATP were 52.3 ± 20.2 μm, 25.2 ± 4.5 μm, 21.7 ± 11.0 μm and 11.6 ± 2.7 μm, respectively. 7 At resting tension, reactive blue 2 (100 μm), a selective antagonist of the P2Y‐purinoceptor, and 8‐(p‐sulphophenyl)‐theophylline (8‐SPT) (1 μm), a selective antagonist of the P1‐purinoceptor, did not antagonize the contractile responses to α,β‐MeATP (0.5–5 μm). Contractile responses to ATP (50–500 μm) were not altered by 8‐SPT (1 μm) but were potentiated by reactive blue 2 (100 μm). 8 With raised tone, ATP and 2‐MeSATP caused a relaxant effect. This effect of ATP was not altered by either tetrodotoxin (TTX) (0.5 μm) or suramin (100 μm), but was antagonized by reactive blue 2 (100 μm) and 8‐SPT (1 μm), suggesting that inhibitory P2Y‐ and P1‐purinoceptors are involved. In contrast, α,β‐MeATP and β,γ‐MeATP caused only contractions. This contractile effect of α,β‐MeATP was resistant to TTX (0.5 μm) and antagonized by suramin (100 μm). 9 In summary, cat colon circular muscle contains postjunctional α1‐adrenoceptors and P2X‐purinoceptors which mediate contractions and P2Y‐ and P1‐purinoceptors which mediate relaxation. Postjunctional α2‐adrenoceptors appear not to be present.

[Met5]enkephalin acts via δ-opioid receptors to inhibit pelvic nerve-evoked contractions of cat distal colon

1 The effects of opioids on the sacral parasympathetic outflow to cat distal colon were studied in vitro using muscle strips orientated in the axis of the longitudinal muscle layer, with pelvic nerves attached. Electrical stimulation of the pelvic nerves evoked contractions that were blocked by atropine (1 × 10−6 m) and tetrodotoxin (3 × 10−7 m). 2 [d‐Pen2, d‐Pen5]enkephalin and [Met5]‐ and [Leu5]enkephalin caused concentration‐dependent, reversible inhibition of pelvic nerve‐evoked contractions, with IC50 values of 8.3 × 10−10 m, 2.2 × 10−9 m and 2.1 × 10−9 m respectively. 3 Morphine (1 × 10−7‐1 × 10−5 m) and [d‐Ala2, MePhe4, Gly‐ol5]enkephalin (1 × 10−8‐1 × 10−6 m) and U‐50,488H (1 × 10−8‐1 × 10−6 m) were much less potent as inhibitors than [Met5]‐ or [Leu5]enke‐phalin. 4 Naloxone (1 × 10−7 m), an antagonist at each of the three opioid receptor types, antagonized the effects of both [Met5]enkephalin and morphine. However, ICI 174,864, a specific δ‐opioid receptor antagonist, antagonised the effects of [Met5]enkephalin only. 5 The inhibitory actions of [Met5]enkephalin were inversely related to frequency of pelvic nerve stimulation. Also, [Met5]enkephalin at a concentration (3 × 10−9 m) which produced a large inhibition of neurogenic contractions, had no effect on contractions to exogenous acetylcholine. These results suggest a prejunctional site for inhibitory opioid receptors. 6 In summary, prejunctional inhibitory δ‐opioid receptors are present on the sacral parasympathetic outflow to cat distal colon; κ‐and/or μ‐opioid receptors may also be present, but appear to be of lesser importance.

Morphology and projections of myenteric neurons to colonic fiber bundles of the cat

Regulation of colon function depends on the location of nerve cell bodies and the distribution of intrinsic nerve fibers in the myenteric plexus. The morphology and projections of myenteric neurons through colonic fiber bundles in cat colon were determined using in vivo retrograde transport of HRP and Fast blue. Myenteric neurons were found to project from at least 5 to 59 mm orad (mean: 42 mm) or aborad (mean: 54 mm) through colonic fiber bundles. Approximately 73% of labelled cells were in ganglia within 2.8 mm of colonic fiber bundles in the axis of circular muscle fibers; none was beyond 7.7 mm. There were 2 soma morphologies. One type (Dogiel type I) had a mean soma diameter of 40.5 microns and had a rough somal surface. There were few if any short, broad dendrites, but its one long process extended to a branch point of an adjacent colonic fiber bundle. The other type (Dogiel type III) had a mean soma diameter of 26.4 microns, had a smooth somal surface and had few if any fine dendrites. It also projected a single long axon to colonic fiber bundles. There were twice as many Dogiel type III neurons. We conclude that myenteric neurons in the cat colon project both orad and aborad over relatively long distances through colonic fiber bundles where they form another intrinsic neuronal connection for the myenteric plexus.

Cholinergic excitatory synaptic potentials of neurones in mammalian lumbar paravertebral ganglia

Synaptic potentials and the electrophysiological properties of 201 cells in the 4th lumbar paravertebral ganglia of the rabbit were studied in vitro using intracellular electrophysiological recording techniques. Cells had a mean transmembrane potential of 55.1 +/- 0.8 mV, a mean input resistance of 37.0 +/- 6.6 M omega (range 29.9-61.1) and a mean membrane time constant of 6.0 +/- 0.6 ms. Synaptic potentials in ganglionic neurones were evoked by electrical stimulation of the rami communicantes, inferior lumbar splanchnic nerves and the paravertebral chain from segments both above and below the L4 ganglion. Synaptic responses consisted of a fast, hexamethonium-sensitive component and, following short periods of higher frequency stimulation, a slow, long lasting, pirenzepine and atropine-sensitive depolarization (slow-EPSP). No phenomenon corresponding to a late slow-EPSP was observed and, under our recording conditions no cells exhibited non-cholinergic slow excitatory or slow inhibitory postsynaptic potentials. It is concluded that fast excitatory synaptic events were mediated by nicotinic receptors whereas slow excitatory synaptic events were mediated by muscarinic m1 receptors. McNeil-A-343, a muscarinic agonist, produced membrane depolarization, a decrease in membrane input conductance and in some cells a repetitive discharge of action potentials. In 60% of cells tested substance P produced a depolarization of the membrane potential with an associated decrease in membrane input conductance.