Thresholds for premature ventricular contractions in frog hearts exposed to lithotripter fields.

Piezoelectrically generated lithotripter shocks were shown to produce premature ventricular contractions of the frog heart. Anesthetized grass frogs, Rana pipiens, were studied following implantation of an aortic catheter and EKG leads. The most sensitive phase of the heart cycle for the generation of premature ventricular contractions with lithotripter shocks at 30 MPa peak pressure was found to be the T-P segment. During this phase of the heart cycle, the minimum peak-positive pressure shock wave necessary to produce a premature ventricular contraction in a frog heart was between 5 MPa and 10 MPa.

The presence and possible role of a galanin-like peptide in the mudpuppy heart.

A correlated histochemical and pharmacological study was undertaken to establish the presence, origin, and possible function of nerve fibers containing a galanin-like peptide in the mudpuppy (Necturus maculosus) heart. Whole mount preparations of septum-sinus venosus or atria and sections of ventricular muscle were prepared for immunocytochemistry. Galanin-immunoreactive fibers were found coursing diffusely across the septum-sinus venosus to form complex networks over cardiac muscle strands. Individual atrial muscle strands were densely innervated by galanin-immunoreactive fibers and galanin-immunoreactive fibers were also observed in the epicardial and myocardial layers of the ventricle. Most of the parasympathetic postganglionic neurons in the cardiac ganglion and many of the small intensely fluorescent-like cells exhibited galanin immunoreactivity. Galanin-immunoreactive fibers were present in the nerve trunks connecting clusters of parasympathetic postganglionic neurons. Close associations between galanin-positive fibers and individual parasympathetic postganglionic neurons were also observed. The presence of the galanin-immunoreactive fibers was similar in preparations taken from animals pretreated with 6-hydroxydopamine to that seen in preparations taken from control animals, indicating that the galanin-positive fibers were not sympathetic postganglionic axons. Moreover, the galanin-immunoreactive nerve fibers were separate from fibers containing substance P and/or calcitonin gene-related peptide that have previously been shown to be processes of afferent fibers. In twitch-tension experiments, galanin in the range 1 x 10(-7) to 1 x 10(-6) M caused cardioinhibition of spontaneously beating isolated septal-sinus venosus preparations. Galanin also produced a concentration-dependent (1 x 10(-7) to 1 x 10(-6) M) decrease in the twitch-tension development of electrically stimulated atrial or ventricular preparations. Local application of galanin produced hyperpolarization of cardiac muscle fibers in both isolated septal-sinus venosus preparations and atrial preparations. The response of individual parasympathetic ganglion cells to local application of galanin varied between neurons; some neurons were depolarized whereas others were hyperpolarized. We conclude that a galanin-like peptide is contained in both the parasympathetic postganglionic neurons and small intensely fluorescent-like cells and their processes. Further, we hypothesize that in the case of the parasympathetic postganglionic neurons, the galanin-like peptide may work in conjunction with acetylcholine to regulate cardiac activity.

Clindamycin-induced alteration of ganglionic function. II. Effect of nicotinic receptor-channel function.

The postsynaptic effects of clindamycin have been analyzed in bullfrog sympathetic ganglion B cells using single electrode current and voltage clamp recordings and two electrode voltage clamp measurements. Clindamycin added to the bathing solution in the concentration range, 2.5 x 10(-4) to 5 x 10(-4) M, inhibited fast ganglionic transmission. In addition, local application of clindamycin decreased depolarizations produced by direct application of acetylcholine and decreased the amplitude of miniature excitatory postsynaptic potentials (MEPSPs) evoked by tetanic stimulation of the preganglionic trunk. In contrast, clindamycin did not change the amplitude or time course of the slow EPSP elicited by preganglionic stimulation (30 Hz for 10 s) or muscarinic depolarizations produced by local acetylcholine application to preparations pretreated with 25-50 microM (+)-tubocurarine. In voltage-clamped ganglion cells, excitatory postsynaptic current (EPSC) amplitude initially was increased and then decreased with increasing concentrations of clindamycin (0.5 x 10(-5) to 2.5 x 10(-4) M). The EPSC time course in control cells was exponential. After exposure to clindamycin, the EPSC decay was composed of two exponential components. The time constant of the fast component decreased and the time constant of the slow component increased with increasing concentrations of clindamycin. The two time constants of EPSCs obtained in clindamycin were independent of membrane voltage between -50 and -100 mV. We concluded that the block of fast ganglionic transmission is primarily due to a postsynaptic site of action, at least part of which is due to a concentration-dependent, but voltage-independent blockade of open nicotinic receptor channel complexes.

Distribution of calcitonin gene-related peptide immunoreactive nerve fibers in the mudpuppy cardiac septum.

An immunohistochemical study was undertaken to determine the distribution of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers in the cardiac septum of the mudpuppy, Necturus maculosus. Numerous long, CGRP-immunoreactive nerve fibers course across the septum, run in the nerve trunks connecting clusters of postganglionic parasympathetic cells, form complexes over groups of ganglion cells and make pericellular networks around individual ganglion cells. The postganglionic parasympathetic neurons and small intensely fluorescent (SIF)-like cells did not exhibit CGRP immunoreactivity. Most of the CGRP-immunoreactive nerve fibers also are labeled for substance P. In freshly dissected preparations, the staining pattern for CGRP was not similar to that obtained using an antiserum against synaptic vesicle membrane, which appears to preferentially label cholinergic preganglionic terminals on all postganglionic parasympathetic cells in the mudpuppy preparation. Further, in explanted ganglia (maintained 10 days in culture) almost no reactivity was obtained with the antivesicle antiserum whereas numerous nerve fibers still exhibited CGRP-immunoreactivity. These observations demonstrate that the CGRP-immunoreactive nerve fibers are not parasympathetic preganglionic axons. Rather we suggest that the CGRP-immunoreactive nerve fibers are processes of primary sensory fibers.

Acceleration of desensitization by agonist pre-treatment in the snake.

1. The kinetics of carbachol-induced desensitization have been studied in snake twitch-muscle fibres maintained in an isotonic potassium propionate solution and voltage clamped to +50 mV. 2. Microperfusion of carbachol (162-756 microM) induces a transient outward current which peaks within a few seconds and then slowly decays towards the base line. The time course of current decay estimates the time course of desensitization onset. 3. Brief exposure (30 s) to a 'conditioning' concentration of agonist (10.8 microM) accelerates the desensitization onset produced by exposure to higher 'test' concentrations of agonist (162-756 microM). 4. The acceleration of desensitization by pre-treatment with 10.8 microM-carbachol was independent of the duration of exposure between 15 and 60 s. This observation indicated that the mechanism responsible for the alteration in desensitization kinetics by treatment with 10.8 microM-carbachol differed from that responsible for the time-dependent development of desensitization produced in the presence of higher carbachol concentrations. 5. Pre-treatment with the muscarinic agonists, methylcholine and bethanechol, did not accelerate 216 microM-carbachol-induced desensitization, suggesting that the alteration of desensitization kinetics by pre-treatment was specific for nicotinic agonists. 6. The conditioning concentrations of carbachol (5.4-10.8 microM) produced no measurable outward current in muscle fibres voltage clamped to +50 mV. Further, in patch-clamp recordings it was observed that, with these concentrations of carbachol, there was no channel activity in many successful patches voltage clamped to +50 mV and, when present, the frequency of channel activity was very low. These results demonstrated that the alteration in desensitization was not the consequence of significant amounts of either receptor activation or desensitization produced by the conditioning concentration. 7. Exposure to 10.8 microM-carbachol for periods of up to 150 s did not change the amplitude of miniature end-plate currents recorded at end-plates voltage clamped to +50 mV. These results also demonstrated that the acceleration of desensitization by pre-treatment with conditioning concentrations of agonist was not due to partial desensitization occurring during the pre-treatment period. 8. Our results are consistent with the view that there are distinct populations of agonist binding sites on the acetylcholine receptor which separately regulate desensitization and channel opening.(ABSTRACT TRUNCATED AT 400 WORDS)

Organization of a vertebrate cardiac ganglion: a correlated biochemical and histochemical study.

A correlated biochemical and histochemical study was undertaken to identify and quantify the presence of different biogenic amines and a substance P-like peptide within the parasympathetic cardiac ganglion of the mudpuppy (Necturus maculosus). Tissue extracts of the cardiac septum containing the parasympathetic cardiac ganglia from control animals were found, by high-pressure liquid chromatography, to contain significant amounts of norepinephrine (NE), epinephrine (E), dopamine (DA), and 5-HT. To allow neural elements of extraganglionic origin to degenerate, ganglia were explanted and maintained in organ culture for 8 d. Extracts from these explanted preparations had no detectable level of E, and NE was reduced, whereas DA and 5-HT levels were similar to those of control preparations. The results indicated that some of the neurons intrinsic to the cardiac septum contain DA and 5-HT and that most (greater than 70%) of the E and NE found in this tissue is of extrinsic origin. Histochemistry of control and explanted preparations showed 5-HT-immunoreactive and catecholamine-containing intrinsic neurons. A substance P-like peptide was identified by radioimmune assay in septal extracts. The peptide content diminished by one-third to one-fifth in preparations maintained in organ culture for 8-14 d, suggesting that a significant amount of the substance P-like peptide is derived from extraganglionic sources. Immunocytochemical studies demonstrated the presence of numerous long substance P-immunoreactive fibers coursing across the septum, branching over cardiac muscle fibers, and forming pericellular networks around individual parasympathetic ganglion cells and clusters of ganglion cells. In addition, numerous small intrinsic neurons exhibited immunoreactivity for substance P. Comparison of the substance P-staining patterns in control and explanted ganglia suggests that the majority of the long substance P-immunoreactive fibers innervating the mudpuppy cardiac ganglion cells are not parasympathetic preganglionic fibers. Rather, it is hypothesized that these fibers are processes of primary sensory fibers. The present observations indicate that the mudpuppy cardiac ganglion exhibits a complex organization similar to that of mammalian sympathetic and enteric ganglia.

Catecholamine, serotonin, and substance P-like peptide containing intrinsic neurons in the mudpuppy parasympathetic cardiac ganglion.

The mudpuppy cardiac ganglion contains 2 neuron types: large parasympathetic postganglionic projection neurons and smaller intrinsic neurons originally described by McMahan and Purves (1976) as intensely fluorescent (SIF) cells. The function of these SIF cells, present in the mudpuppy cardiac ganglion, is unknown. Further, direct application of catecholamines, which are thought to be contained in SIF cells, to the parasympathetic postganglionic cells has no effect (Hartzell et al., 1977). As SIF cells in other ganglion preparations recently have been shown to contain putative transmitter substances in addition to catecholamines, immunocytochemical experiments were conducted to test for the presence of additional transmitter substances in the SIF cells within the cardiac ganglion. Whole-mount septal preparations were dissected from Necturus maculosus and processed for indirect immunocytochemistry. The results indicated that many of these intrinsic neurons contained 5-HT or a substance P-like peptide, or both. Many small intrinsic neurons which contain either substance P or 5-HT were also positive for aqueous-aldehyde-induced fluorescence, indicating the presence of a catecholamine. Finally, some of these cells appeared to contain all 3: a catecholamine, 5-HT, and a substance P-like peptide.

Anatomical evidence for the interaction of sympathetic and parasympathetic neurons in the cardiac ganglion of Necturus.

Evidence of a direct interaction between sympathetic and parasympathetic elements in a cardiac parasympathetic ganglion is presented in this study. Experiments were done using acutely dissected or organ cultured parasympathetic cardiac ganglion preparations from Necturus maculosus (mudpuppy). The glyoxylic acid-induced fluorescence technique was used to visualize catecholamine-containing cells and fibers. Numerous long brightly fluorescent varicose fibers form a complex network over clusters of parasympathetic ganglion cells and strands of cardiac muscle. In addition to these fibers, there are numerous small brightly fluorescent interneurons (SIF cells) interspersed between individual parasympathetic ganglion cells. Long fibers and processes from the interneurons join to form bundles which arborize over groups of parasympathetic cells. In peripherally located smaller groups of ganglion cells there are no interneurons, but some of these parasympathetic cells appear to receive innervation from the long continuous fluorescent axons. Two experimental procedures were applied to support the conclusion that these long fibers were indeed sympathetic postganglionic axons: explants of cardiac ganglia were maintained for varying times to produce degeneration of any severed axons: chemical sympathectomy was produced by injection of 6-hydroxydopamine. The intrinsic SIF cells were apparently unaffected by both procedures. After 8 days in culture or after 6-OH dopamine treatment, all of the long continuous brightly fluorescent fibers, which normally intermingle with clusters of ganglion cells or innervate cardiac muscle, were absent. This indicates their extra-ganglionic origin. All of the isolated groups of parasympathetic ganglion cells not containing SIF cells were totally devoid of any catecholamine-containing fibers.

Influence of the extracellular ionic environment on ganglionic fast excitatory postsynaptic currents.

The influence of changing the extracellular sodium or calcium concentration as well as the addition of strontium or magnesium on characteristics of nicotinic fast excitatory postsynaptic currents (EPSCs) has been studied in voltage-clamped sympathetic ganglion B cells of the bullfrog. Lithium substitution for sodium influenced both the EPSC size and decay time course in a concentration-dependent manner. In those cells exposed to either a 50% or 100% lithium-substituted solution, the EPSC decay was faster than that of control EPSCs. With a 50% replacement of lithium for sodium, the EPSC size at -520 mV was similar to control values. However, with a 100% substitution, the EPSC size was significantly reduced below control values although the voltage dependence of the decay tau, the shape of the peak EPSC-voltage relationship, or the EPSC reversal potential was not changed by replacing lithium for sodium. The change in EPSC size and decay tau in the lithium solution was due to the presence of lithium and not simply the consequence of a reduction in the external sodium concentration; as with a 50% substitution of sucrose or mannitol for sodium chloride the EPSC decay was slowed. EPSC size at -50 mV and the voltage dependence of tau was similar to control values when 50% of the sodium was replaced by sucrose. The peak EPSC-voltage relationship was linear in cells exposed to either the control or the 50% sucrose-substituted solution, although the EPSC reversal potential was shifted to a more negative voltage with 50% sucrose substitution.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of thyroid hormone levels on the electrical and mechanical properties of rabbit papillary muscle.

We have examined the influence of chronic in vivo alterations of thyroxine levels on the electrophysiological and mechanical properties of rabbit ventricular papillary muscles measured in vitro. Marked changes in the repolarization phase of the action potential and the time to peak tension of isometric twitches were observed when thyroid hormone levels were increased above or decreased below normal. The time to peak tension was consistently shorter than normal in hyperthyroid and longer than normal in hypothyroid preparations. In hypothyroid preparations the action potential duration was greater than that of controls at all stimulation frequencies tested (0.1 to 1.0 Hz). In hyperthyroid preparations, the repolarization phase consisted of an initial phase of fast repolarization to membrane potential values between -20 and -40 mV followed by a plateau. The early phase of repolarization persisted at all stimulation frequencies tested (0.1 to 1.0 Hz) but the plateau component increased markedly as the stimulation frequency was decreased. The early phase of repolarization was markedly reduced in the presence of 4-aminopyridine. These results suggest that thyroxine levels may modulate the kinetics of a transient outward current which in rabbit papillary muscle normally is responsible for the frequency dependence of action potential duration. Action potential amplitude, maximum rate of rise, resting membrane potential, and peak isometric twitch tension were not markedly different between the three classes of preparations. A second depolarizing response occurred in all hyperthyroid preparations at low stimulation frequencies (0.1 to 0.4 Hz) but not in control or hypothyroid preparations. This second depolarizing response, which was eliminated by D-600 treatment, was similar to the calcium-dependent slow action potentials recorded in other cardiac preparations. These two component action potentials could represent either intrinsic single cell activity, or a re-entry wave of depolarization which results from nonhomogeneous excitation.

Comparison of cholinergic activation and desensitization at snake twitch and slow muscle fibre end-plates.

Characteristics of receptor-channel activation and desensitization have been compared at voltage-clamped snake slow and twitch fibre end-plates maintained in an isotonic potassium propionate solution. Miniature end-plate current (m.e.p.c.) decay was slower and less voltage dependent at slow fibre end-plates than at twitch fibre end-plates. The peak m.e.p.c. amplitude versus voltage relationship and reversal potential were similar at the two end-plate types. Acetylcholine-induced noise and m.e.p.c.s were recorded at slow fibre end-plates. At most slow fibres the spectral density was not adequately fitted by a single Lorentzian function. Rather, the observed spectral density was greater at high frequencies than the values predicted using the m.e.p.c. decay rate. The noise could be well described by the sum of two Lorentzian functions, one of which corresponded to a single Lorentzian function with the corner frequency determined by the m.e.p.c. decay rate. The shape of the carbachol concentration-peak end-plate current relationship was similar at both slow and twitch fibre end-plates. However, for all concentrations tested, the peak carbachol-induced end-plate current (e.p.c.carb.) value was markedly less at slow fibre end-plates than at twitch fibre end-plates. The onset of desensitization was determined using two methods. The first concerned analysis of the time course of decay of the e.p.c.carb. from a peak value during the sustained application of agonist. The second involved a double-perfusion technique in which a 'desensitizing' dose was applied for varying intervals before the application of a second 'test' dose of carbachol. With both methods the development of desensitization at both end-plate types was dependent on carbachol concentration and duration of exposure. At each end-plate type the time course of desensitization onset often exhibited two components; one with a time constant of seconds and a slower component having time constants in the range of tens to hundreds of seconds. The slope of the relationship between carbachol concentration and equilibrium desensitization at slow and twitch fibre end-plates was close to two, suggesting that two molecules of agonist are probably bound during the development of desensitization. However, for all concentrations tested, desensitization developed more rapidly and to a greater extent at twitch fibre end-plates than at slow fibre end-plates. The voltage dependence of the 3 min steady-state desensitization produced by 108 microM-carbachol was very similar (approximately -0.0250 mV-1) at both fibre types.(ABSTRACT TRUNCATED AT 400 WORDS)