Active Membrane Responses Research Articles

Evidence against a Role of Elevated Intracellular Ca2+ during Plasmodium falciparum Preinvasion

Severe malaria is primarily caused by Plasmodium falciparum parasites during their asexual reproduction cycle within red blood cells. One of the least understood stages in this cycle is the brief preinvasion period during which merozoite-red cell contacts lead to apical alignment of the merozoite in readiness for penetration, a stage of major relevance in the control of invasion efficiency. Red blood cell deformations associated with this process were suggested to be active plasma membrane responses mediated by transients of elevated intracellular calcium. Few studies have addressed this hypothesis because of technical challenges, and the results remained inconclusive. Here, Fluo-4 was used as a fluorescent calcium indicator with optimized protocols to investigate the distribution of the dye in red blood cell populations used as P. falciparum invasion targets in egress-invasion assays. Preinvasion dynamics was observed simultaneously under bright-field and fluorescence microscopy by recording egress-invasion events. All the egress-invasion sequences showed red blood cell deformations of varied intensities during the preinvasion period and the echinocytic changes that follow during invasion. Intraerythrocytic calcium signals were absent throughout this interval in over half the records and totally absent during the preinvasion period, regardless of deformation strength. When present, calcium signals were of a punctate modality, initiated within merozoites already poised for invasion. These results argue against a role of elevated intracellular calcium during the preinvasion stage. We suggest an alternative mechanism of merozoite-induced preinvasion deformations based on passive red cell responses to transient agonist-receptor interactions associated with the formation of adhesive coat filaments.

Neurogliaform cells of amygdala: a source of slow phasic inhibition in the basolateral complex

Synaptic inhibition in the amygdala actively participates in processing emotional information. To improve the understanding of interneurons in amygdala networks it is necessary to characterize the GABAergic cell types, their connectivity and physiological roles. We used a mouse line expressing a green fluorescent protein (GFP) under the neuropeptide Y (NPY) promoter. Paired recordings between presynaptic NPY-GFP-expressing (+) cells and postsynaptic principal neurons (PNs) of the basolateral amygdala (BLA) were performed. The NPY-GFP+ neurons displayed small somata and short dendrites embedded in a cloud of highly arborized axon, suggesting a neurogliaform cell (NGFC) type. We discovered that a NPY-GFP+ cell evoked a GABA(A) receptor-mediated slow inhibitory postsynaptic current (IPSC) in a PN and an autaptic IPSC. The slow kinetics of these IPSCs was likely caused by the low concentration and spillover of extracellular GABA. We also report that NGFCs of the BLA fired action potentials phase-locked to hippocampal theta oscillations in anaesthetized rats. When this firing was re-played in NPY+-NGFCs in vitro, it evoked a transient depression of the IPSCs. Presynaptic GABA(B) receptors and functional depletion of synaptic vesicles determined this short-term plasticity. Synaptic contacts made by recorded NGFCs showed close appositions, and rarely identifiable classical synaptic structures. Thus, we report here a novel interneuron type of the amygdala that generates volume transmission of GABA. The peculiar functional mode of NGFCs makes them unique amongst all GABAergic cell types of the amygdala identified so far.

Functional connectivity of the main intercalated nucleus of the mouse amygdala

Intercalated cells (ITCs) of the amygdala are clusters of GABAergic cells that surround the basolateral complex of the amygdala (BLA). Growing evidence suggests that ITCs are required for the expression of fear extinction. The main intercalated nucleus (Im) is the largest of the ITC clusters and could also be important for emotional processing. We used whole-cell recordings from Im neurons in acute slices of mouse amygdala. We found that these neurons were medium-sized spiny projection cells. Their passive and active membrane responses were consistent with those previously reported in other ITC clusters. The axon of Im neurons was, in many cases, cut at the slice boundaries, suggesting long-range projections. Axonal branches could be detected in several amygdala nuclei where they made functional synapses. We also functionally studied Im cell inputs. Excitatory postsynaptic currents (eEPSCs) were evoked by the stimulation of the Im, intermediate capsula (IC), external capsula (EC) or BLA, when GABAergic transmission was pharmacologically blocked. An occlusion test indicated that fibres recruited by stimulating Im and IC, or Im and EC were distinct. These eEPSCs had both NMDA and AMPA receptor components. Inhibitory postsynaptic currents (eIPSCs) were evoked after the stimulation of the Im, the EC and the BLA, when glutamatergic transmission was pharmacologically blocked. Furthermore, dopamine reversibly hyperpolarised, and decreased the firing frequency and the input resistance of Im cells via dopamine type 1 receptor. Our data suggest that the Im is functionally connected to other amygdala nuclei and is under neuromodulatory influence. We propose that the Im serves as key neuronal substrate of fear extinction.

The role of main intercalated nucleus of the mouse amygdala

Event Abstract Back to Event The role of main intercalated nucleus of the mouse amygdala M. Manko1*, R. Geracitano1 and M. Capogna1 1 Anatomical Neuropharmacology Unit, United Kingdom Intercalated cell masses (ITC) of the amygdala are clusters of GABAergic cells that surround the basolateral complex of the amygdala (BLA). There is growing evidence suggesting that ITCs contributes to fear expression and memory1. Our study aims to characterize for the first time the main intercalated nucleus (Im), the largest of the ITCs cluster, located ventrally to the BLA. We studied in acute mice slices the electrophysiological responses of anatomically identified neurons of the Im and the effects of dopamine, since this neuromodulator gates fear learning2. We found that the Im cluster consists of medium- sized, spiny neurons with bipolar dendritic organization and with axon that project to the majority of other amygdala nuclei and the intermediate capsula (IC). Whole cell patch clamp recordings (n=50) show passive and active membrane responses consistent with those previously detected in other clusters of ITCs3,4. We also functionally studied Im cells’ inputs/outputs. Monosynaptic excitatory postsynaptic currents were observed when we stimulated extracellularly the Im (n=6/6), the intermediate capsula (n=2/6), the external capsula (EC, n=3/6), or the BLA (n=2/6). An occlusion test indicated that fibres recruited by stimulating Im and IC, or Im and EC are distinct. Monosynaptic inhibitory postsynaptic currents were always observed after stimulation of the Im (n=6/6), but also of the IC (n=2/7) or EC (n=3/7), indicating functional connectivity among ITC clusters. Furthermore, dopamine (30 microMol) significantly hyperpolarised and decreased the firing frequency of Im cells (n=11) in a reversible manner. Thus, we propose that the Im cells, and not only mITCs, may be a key neuronal substrate of fear extinction.

The Edinger-Westphal nucleus of the juvenile rat contains transient- and repetitive-firing neurons

Classically, the Edinger-Westphal nucleus is described as containing neurons controlling accommodation and pupillary constriction via projections to the ciliary ganglion. However, in several species including rat, some Edinger-Westphal neurons have ascending or descending CNS projections suggesting that the Edinger-Westphal nucleus might also have non-ocular functions. To further characterize the function of this nucleus we studied the electrophysiological properties of Edinger-Westphal neurons in a slice preparation from juvenile rats. The position of the Edinger-Westphal nucleus was determined using an immunohistochemical procedure directed at the peptide Urocortin, which is expressed in Edinger-Westphal neurons. Passive and active membrane responses were investigated and two different neuron types were identified. One type had a transient firing response to 400 ms depolarizing current pulses and one type had a repetitive firing response. Transient-firing neurons had an outward rectifying response inhibiting firing, possibly due to slowly inactivating ID-like potassium channels since low concentrations (200 μM) of the potassium channel blocker 4-aminopyridine elicited repetitive firing. In all neurons, low threshold Ca2+ spikes were seen and these were blocked by nickel(II) chloride hexahydrate, suggesting that they are mediated via low voltage–activated Ca2+ channels. Some biocytin-labeled neurons had axons or axonal collaterals projecting laterally or dorsally, suggesting possible non-ocular targets. In conclusion, the rat Edinger-Westphal nucleus contains two separate types of neurons with distinct electrophysiological properties.

Electrophysiological properties of cultured hippocampal neurons from Wistar Audiogenic Rats

The main goal of this work was to analyze the electrophysiological properties of cultured hippocampal neurons from a particular epileptic rat strain, called Wistar Audiogenic Rats (WAR). The whole-cell patch-clamp technique was used to record both active and passive membrane responses in an attempt to detect alterations in their characteristics in relation to controls from Wistar rats. Neurons from WARs show a significant reduction in the magnitude of the inhibitory GABAergic currents (∼45%), in spite of maintaining a normal level of the excitatory glutamatergic currents. In addition, the magnitude of potassium currents, measured at +80 mV, is reduced by about 30% in comparison to controls. Surprisingly, we also found important changes in the passive cellular properties in WAR neurons such as membrane potential (−50.0 mV in WARs and −63.1 mV in controls) and input resistance (647 MΩ in WARs and 408 MΩ in controls). The changes described here, could be the basis of the neurophysiological and behavioral alterations present in these hyperexcitable animals, contributing to a better understanding of epileptogenesis in this particular animal model.

The modulation of skeletal muscle contraction by FMRFamide-related peptides of the locust

The external ventral protractor muscle of the VIIth abdominal segment, M234, is a skeletal muscle that possesses receptors that recognize a range of FMRFamide-related peptides and application of these peptides results in an increase in the amplitude of neurally evoked contractions with little or no effect on basal tonus. FLRFamide itself has the same biologic activity as the extended peptides, whereas truncation to LRFamide or RFamide results in a peptide with no biologic activity. The receptors recognizing these extended FLRFamides, which include SchistoFLRFamide, seem to be different from the SchistoFLRFamide receptors found on locust oviduct visceral muscle. SchistoFLRFamide and the non-peptide mimetic, benzethonium chloride (Bztc), increase the frequency and amplitude of miniature endplate potentials, increase the amplitude of neurally evoked excitatory junction potentials, and result in a hyperpolarisation of resting membrane potential. Bztc, however, also abolishes the active membrane response that may explain its ability to decrease neurally evoked contractions.

Regional differences in sympathetic neurotransmission to cutaneous arteries in the guinea-pig isolated ear

The effects of sympathetic nerve stimulation on different cutaneous arteries were examined in arteries isolated from guinea-pig ears, by measuring membrane potential changes in smooth muscle cells in response to electrical field stimulation. Resting membrane potential (RMP) was similar in proximal (main ear artery) and distal (3rd or 4th branch order) cutaneous arteries (mean −71 mV). Single stimuli evoked excitatory junction potentials (EJPs) in all arteries. The EJPs in proximal arteries were twice the amplitude, and the time constant of EJP decay was almost half the value, compared with distal cutaneous arteries. EJP amplitude was reduced by >90% by suramin (30 μM) or α,β,methylene-ATP (α,β,m-ATP) (1 μM) in all proximal, and most distal arteries. Residual responses in distal arteries were resistant to tetrodotoxin. The N-type calcium channel blocker, ω-conotoxin GVIA (30 nM), reduced EJP amplitude by 70–100% in both proximal and distal arteries. Successive EJPs evoked by trains of stimuli at 1 to 5 Hz were depressed in amplitude in proximal arteries, but showed facilitation in distal arteries. EJP depression in proximal arteries was reversed to facilitation by the α 2-adrenoceptor antagonist, yohimbine (30 nM). Trains of stimuli delivered at 10–20 Hz produced summation of EJPs and active membrane responses in 30% of proximal arteries. Active responses were never detected in distal arteries. Slow depolarizations following the EJPs were detected in most arteries after trains of stimuli, and were abolished by prazosin (0.3 μM) or ω-conotoxin GVIA (30 nM). The density of the perivascular plexus of axons innervating proximal arteries, demonstrated with catecholamine fluorescence histochemistry, was twice that in distal cutaneous arteries. These regional differences in sympathetic neurotransmission suggest that cutaneous vasoconstriction in response to thermoregulatory stimuli, which occurs predominantly in distal cutaneous segments, is likely to be qualitatively different from cutaneous vasoconstriction of proximal arteries in response to other physiological stimuli.

Modification of discharge patterns of neocortical neurons by induced oscillations of the membrane potential

We investigated, with whole-cell recordings from rat visual cortex slices, how sinusoidal modulation of the membrane potential affects signal transmission. Subthreshold oscillations activate tetrodotoxin sensitive, transient inward currents whose threshold, phase lag and duration change with modulation frequency. These periodically recurring phases of enhanced excitability affect synaptic transmission in two ways. Weak and short lasting excitatory postsynaptic potentials evoke discharges only if they are coincident within a few milliseconds with these active membrane responses. Long-lasting, N-methyl- d-aspartate-mediated or postsynaptic excitatory postsynaptic potentials, by contrast, evoke trains of spikes, that are precisely time-locked to the oscillations and may last for more than 100 ms. Thus, oscillations impose a precise temporal window for the integration of synaptic inputs, favouring coincidence detection and they generate temporally-structured responses whose timing and amplitude are largely independent of the input. These properties are ideally suited for the synchronization of neuronal activity and the encoding of information in the precise timing of discharges. A preliminary account of these data has appeared in an abstract form [Volgushev M. et al. (1995) Eur. J. Neurosci. 8, 77].

Interaction between afferent neurones in a crab muscle receptor organ

Two of the three mechanoreceptor neurones which innervate the crab thoracic-coxal muscle receptor organ are non-spiking. The third, the small diameter P neurone, employs graded amplitude receptor potentials with either spikes or graded amplitude active membrane responses superimposed. An excitatory synaptoc connection between one of the larger non-spiking afferents and the P neurone has now been detected within the thoracic ganglion. This novel connection between two afferents innervating the same receptor strand effectively extends the dynamic sensitivity range of the P neurone.

Action potentials in a ‘non-spiking’ neurone: graded responses and spikes in the afferent P fibre of the crab thoracic-coxal muscle receptor organ

The small diameter P neurone (7 μm) is here characterized as an afferent of the crab thoracic-coxal muscle receptor organ (TCMRO), in addition to the S and T neurones (50–60 μm). The P neurone employs both passively conducted, graded amplitude receptor potentials and, superimposed upon these, either graded active membrane responses or constant amplitude action potentials. Intracellularly recorded receptor potential waveforms reflect the dynamic component of stretch more strongly than receptor length, while action potentials at the onset of stretch accentuate the phasic response characteristics of this neurone.

The electrical and mechanical properties of the proleg retractor muscle of the Chinese oak silkmoth larva

ABSTRACT. The main proleg retractor muscle (y) of Antheraea pernyi Guer. (Lepidoptera, Saturniidae) larvae consists of three layers of fibres. The innermost layer of fibres is dually innervated. Cobalt backfills of the two motor neurones, in nerve 2d, showed the somata to be situated ventrally and anteriorly in the same segmental ganglion, ipsilateral to the filled nerve. Intramuscular microelectrode recordings showed excitatory junction potentials (EJPs) of two distinct amplitudes, both of which were relatively slow. However, 26% of the larger amplitude EJPs had an active membrane response. The EJPs and mechanical responses both summated at low stimulation frequencies. Large EJPs resulted in a much greater development of tension than small ones. Extracellular stimulation of nerve lbiii modulated peak tension and peak rate of relaxation.

Passive and active membrane properties of mudpuppy taste receptor cells.

1. Intracellular recordings were obtained from taste receptor cells and surface epithelial cells of isolated mudpuppy lingual epithelium. 2. Surface epithelial cells had a mean resting potential of -40.2 +/- 8.9 mV, a mean input resistance of 40.3 +/- 11.3 M omega, and a linear current-voltage (I-V) relationship. Taste receptor cells had a mean resting potential of -61.7 +/- 15 mV, a mean input resistance of 380.3 +/- 177.2 M omega, and the I-V relationship showed pronounced outward rectification; the outward rectification persisted in high-K+ saline, but was abolished by tetraethylammonium bromide (TEA). 3. Surface epithelial cells responded to depolarizing current injection with only passive membrane potential changes. Taste receptor cells responded to brief pulses of depolarizing current injection with regenerative action potentials characterized by an abrupt rising phase, an inflexion on the falling phase, and a prolonged after-potential. 4. The abrupt rising phase of the action potential was blocked by tetrodotoxin (TTX), suggesting that voltage-gated Na+ currents are responsible for the rising phase. 5. Long-duration action potentials were elicited from cells treated with TEA to block outward K+ currents and with TTX to block Na+ currents, and from cells bathed in isotonic CaCl2. These results suggest that the active membrane response contains a significant Ca2+ component. 6. The after-potential was blocked or greatly reduced by the addition of Ca2+ channel blockers to the bathing medium. In contrast, addition of TEA to the bathing medium greatly enhanced the after-potential. These data suggest that a significant portion of the after-potential is Ca2+ mediated. 7. The mean reversal potential for the after-potential (-76.8 +/- 6.0 mV) was significantly different from the mean reversal potential for the undershoot of the action potential (-86 +/- 5.6 mV). Superfusion with TEA reduced the reversal potential of the after-potential to -42.3 +/- 8.2 mV and abolished the undershoot. These results suggest that the after-potential results from at least two conductances, one which is blocked by TEA and the other which is Ca2+ dependent and involves ions other than, or in addition to K+. 8. Our data suggest that taste receptor cells, unlike surface epithelial cells, possess voltage-gated Na+, Ca2+, and K+ channels, as well as Ca2+-mediated channels. The role of the Ca2+ channels may be in part to regulate release of transmitter onto nerve terminals. The role of the other conductances in taste transduction is unknown.

Endogenous photoproteins, calcium channels and calcium transients during metamorphosis in hydrozoans.

There are species of hydrozoans, Eutonina victoria, Mitrocomella polydiademata, and Phialidium gregarium whose eggs contain calcium-specific photoproteins. These cytoplasmic photoproteins are synthesized during oogenesis. During the cleavage stages of embryogenesis they are distributed to all of the cells of the developing planula larva. The amount of photoprotein slowly declines during the development of the planula larva, and markedly declines when the planula undergoes metamorphosis to become a polyp.Oocytes, unfertilized eggs, and fertilized eggs prior to the first cleavage do not produce light when treated with KCl. The ability to respond to KCl appears about the time of first cleavage, and is correlated with the appearance of active membrane responses. Both the KCl response and the action potentials will occur in sodium-free sea water, and both are inhibited by calcium channel blockers. These and other experiments suggest that voltage sensitive calcium channels first become active at about the time of first cleavage. These channels also appear on the same schedule in both unfertilized eggs and in enucleated egg fragments, which have been artificially activated with A23187.Developing planulae produce few or no spontaneous light responses before gastrulation. Later the frequency and magnitude of spontaneous light production increases presumably due to an increasing frequency and magnitude of calcium transients. Both the natural trigger of metamorphosis (bacteria) and an artificial trigger (CsCl) cause a conspicuous series of calcium transients. When these transients are inhibited by calcium channel blockers, metamorphosis is also inhibited.

Octopamine enhances neuromuscular transmission in developing and adult moths, Manduca sexta.

The effect of octopamine on neuromuscular transmission was examined in developing and adult Manduca sexta. Intracellular recordings were made from the dorsal longitudinal muscle (DLM), superfused with solutions containing DL-octopamine or other amines. In untreated adult moths and pharate adults nearly ready to enclose (stage Day 19), stimulation of the motor nerve evokes a large excitatory junction potential (EJP), an active membrane response, and a twitch. In adults and Day 19 animals DL-octopamine (10(-7) to 10(-4)M) has no effect on the amplitude and rise-time of the electrical response in normal saline, but 10(-6) to 10(-4) M DL-octopamine increases the amplitude of the excitatory junction potential recorded in saline containing one-third the normal calcium concentration. Immature (Day 16) muscle, which normally produces only small EJPs following stimulation of its motor nerve, responds to 10(-6) to 10(-4) M DL-octopamine by an increase in the EJP above threshold for an active membrane response and a contraction. When the muscle has developed sufficiently to spike and contract in response to nerve stimulation in the absence of exogenous octopamine (Days 17 and 18), application of DL-octopamine increases the maximum rate at which the muscle contracts in response to each stimulus in a train (designated the maximum following frequency, MFF). The threshold dose for an effect on the MFF of Day 18 immature moths is less than 10(-10) M. At this stage 10(-8) M DL-octopamine increases the MFF four-fold. The effect on the MFF is dose-dependent over the range 10(-10) M to 10(-6) M. The biogenic amines DL-epinephrine, DL-norepinephrine, tyramine, DL-phenylethanolamine, 2-phenylethylamine, and dopamine, applied at concentrations of 10(-8) or 10(-4) M, do not change the MFF. Both DL-synephrine (10(-8) M) and serotonin (10(-7) M) mimic the action of 10(-10) M DL-octopamine on the MFF. The action of DL-octopamine (10(-7) M) is blocked by phentolamine (10(-4)M) but not by propranolol (10(-4)M). The octopamine content of hemolymph was determined with a radioenzymtic assay. The concentration of octopamine in the hemolymph increases 3.6-fold, from 5 X 10(-8) M on Day 18 (duration of adult development is 19 days) to 1.85 X 10(-7) M one day following eclosion.(ABSTRACT TRUNCATED AT 400 WORDS)

Giant synaptosomes.

Investigations using synaptosomes, pinched-off nerve ending particles from brain, have greatly improved our knowledge of presynaptic function. However, these structures, like most nerve endings, are too small to be penetrated with microelectrodes. We have treated synaptosomal preparations from rat brain with a neutral protease and obtained fused structures large enough to be recorded from directly with microelectrodes; we report here that these particles (30-250 microM in diameter) contain mitochondria and structures resembling synaptic vesicles, morphological features characteristic of synaptosomes. These 'giant synaptosomes' have resting membrane potentials in the range -45 to -76 mV, are depolarized by increasing concentrations of K+, show responses to a variety of neuroactive substances and exhibit active membrane responses to depolarizing current pulses. These results suggest that this preparation will be of value in further studies of nerve terminal function.

Neurophysiology of adult male Schistosoma mansoni

From the studies on the neurophysiology of schistosomes it appears that in spite of the unique hexilaminar arrangement of the tegument's outer membrane, it has biophysical properties not markedly different from those of a variety of other multi-dimensional syncytia. The close electrical coupling of the muscle and tegument must be taken into account when one attempts to define sites and modes of action of drugs which affect motor activity. Agents which disrupt muscle function in the schistosome may exert their action indirectly by way of an effect on the tegumental membrane. The syncytial nature of the musculature and the possibility that longitudinal contraction waves are myogenic suggests that neurotransmitters may simply function as modulators of muscle activity as is the case for many vertebrate visceral muscles. External recordings indicate the presence of a variety of electrically active tissues within the schistosomes. There is no clear correlation of this activity with longitudinal muscle activity or with active membrane responses in this muscle. From this it would appear the bulk of this activity may have its origins in tissues other than the longitudinal muscle such as other muscle groups, nerve trunks, or the peripheral nerve net.

Microelectrode studies of the tegument and sub-tegumental compartments of male Schistosoma mansoni: an analysis of electrophysiological properties.

Standard intracellular microelectrode techniques were used to determine the electrical properties of the tegument and sub-tegumental regions in male Schistosoma mansoni. Three distinct compartments of electrical potential were observed. The resting potentials recovered in these compartments of -45.9 +/- 2.5 mV (Eteg), -22.0 +/- 1.1 mV (E2) and -4.7 +/- 03 mV (E3) corroborate those previously reported by Fetterer, Pax & Bennett (1980) and Bricker, Pax & Bennett (1981). Input resistance was measured in each compartment and was found to be 4. 5 M omega (tegument), 9.2 M omega (E2) and 3.5 M omega (E3). Time-constants for the tegument, E2 and E3 were 0.24 +/- 0.01 msec, 0.25 +/- 0.01 msec and 0.13 +/- 0.01 msec, respectively. Multiple electrode experiments revealed that the tegument and E2 compartment are electrical syncytia with similar current-spreading capabilities. Low resistance pathways also appear to connect the tegument and E2 region, since electrotonic signals initiated in either of those compartments experience only a 15-25% reduction upon passing into the other. Injecting large (greater than 200 nA) depolarizing current pulses into the tegument or E2 compartment often resulted in the initiation of active membrane responses. These spikes were highly variable, ranging from 4 to 75 mV in magnitude (occasionally overshooting zero potential by as much as 25 mV) and from 10-40 msec in duration. The responses were not actively propagated along the parasite, and their decay over distance was approximately equal to that predicted on the basis of length constant values obtained from electronic signals. The addition of a non-diffusible solute to the recording medium resulted in a significant reduction in the current-spreading capacity of both the tegument and E2 compartment. Coupling ratios between the tegument and E2 compartment. Coupling ratios between the tegument and E2 compartment were decreased, and the input resistance for both compartments increased, while resting potentials remained constant. Active responses could not be evoked in schistosomes exposed to the hyperosmotic medium.

Intracellular Recording of Electrical Activity in Muscle Cells of Intact and Isolated Dorsal Blood Vessel of the Earthworm Lumbricus Terrestris

ABSTRACT Intracellular electrical records from muscle cells in intact dorsal blood vessels of the earthworm, Lumbricus terrestris, showed a polarization rhythm which consisted of : (a) a prolonged ramp of depolarization, (b) one or a series of several non-overshooting spike potentials, (c) a sustained, hump-like depolarization, and (d) a complete repolarization. The polarization rhythm of the intact vessel was reset by intracellular current injection. Simultaneous recordings of intracellular electrical and mechanical activities during rhythmic pulsation waves of the intact vessel indicated that the ramp-like depolarization corresponded temporally to a gradual filling of the vessel with blood. Spike potentials occurred during the later portion of filling, and constriction began near the peak of the slow, sustained depolarization. Simultaneous intracellular and extracellular electrical records during rhythmic pulsations of the intact vessel indicated a close temporal correspondence between spike potentials in both electrical records. The repolarization phase in intracellular records corresponded to a small, slow wave in extracellular records. A polarization rhythm was usually absent in isolated preparations of the dorsal blood vessel, but in a few preparations rhythmic activity persisted for up to 1 h. In quiescent isolated preparations membrane potentials were stable (mean resting potential = −66·7 mV), but active membrane responses (non-overshooting spikes and sustained depolarizations), were evoked by intracellular current injection or by stretching of the vessel. Evidence from dual intracellular recordings in isolated dorsal vessels suggest that muscle cells are electrically coupled. The intracellular activity patterns in intact and isolated vessels, and the responsiveness of cells to current injection and stretching, suggest that both initiation and conduction of pulsation waves in the dorsal blood vessel are myogenic.

Angiotensin induction of active responses in cultured reaggregates of rat aortic smooth muscle cells.

Electrical activity was studied in cultured reaggregates of vascular smooth muscle cells prepared from the aortas of normotensive (WKY) and spontaneously hypertensive (SHR) rats. There were no significant differences between the resting membrane potentials of WKY (- 44.7 +/- 0.5 mV) and SHR (- 45.6 +/- 0.5 mV) reaggregates. Electrical field stimulation elicited active responses that consisted of two components: an initial spike component (amplitude, 30-40 mV; +Vmax 1-3 V/s) and a plateau component (amplitude, 10-30 mV; duration 3-5s; +Vmax, 0.1-0.3 V/s). Often the spike component was absent. Both components of the response were abolished by verapamil, Mn++, and Na+-free solutions indicating that Na+ and Ca++ currents may be associated with them. Addition of 10 mM TEA increased the duration of the response, and addition of 0.5 mM BaCl2 to the TEA solution increased the duration even further. The spike component always occurred in the presence of TEA and Ba++. No differences were observed between responses in WKY and SHR reaggregates. A bolus of angiotensin II (AII) (bolus concentration of 1 micro M) elicited a transient depolarization. In some cells an active membrane response was triggered by the depolarization and sometimes included the spike component. Replacing all the Na+ in the solution with either Tris or choline resulted in loss of the response to AII, and in some cells a hyperpolarization was actually seen on addition of the AII. The response to AII was insensitive to verapamil (up to 10(-5) M), but was blocked by 1 mM MnCl2. Reaggregates from both WKY and SHR animals gave similar responses to AII. The data indicates that cultured reaggregates of vascular smooth muscle are electrically excitable and are sensitive to angiotensin. Therefore functional angiotensin receptors must be present in the cultured cells.